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Accelerated Response of the myogenin Gene to Denervation in Mutant Mice Lacking Phosphorylation of Myogenin at Threonine 87

Chris S. Blagden, Larry Fromm, and Steven J. Burden^*

Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, New York University Medical School, New York, New York 10016

Received 24 October 2003/ Returned for modification 23 November 2003/ Accepted 3 December 2003

	ABSTRACT

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Gene expression in skeletal muscle is regulated by a family of myogenic basic helix-loop-helix (bHLH) proteins. The binding of these bHLH proteins, notably MyoD and myogenin, to E-boxes in their own regulatory regions is blocked by protein kinase C (PKC)-mediated phosphorylation of a single threonine residue in their basic region. Because electrical stimulation increases PKC activity in skeletal muscle, these data have led to an attractive model suggesting that electrical activity suppresses gene expression by stimulating phosphorylation of this critical threonine residue in myogenic bHLH proteins. We show that electrical activity stimulates phosphorylation of myogenin at threonine 87 (T87) in vivo and that calmodulin-dependent kinase II (CaMKII), as well as PKC, catalyzes this reaction in vitro. We find that phosphorylation of myogenin at T87 is dispensable for skeletal muscle development. We show, however, that the decrease in myogenin (myg) expression following innervation is delayed and that the increase in expression following denervation is accelerated in mutant mice lacking phosphorylation of myogenin at T87. These data indicate that two distinct innervation-dependent mechanisms restrain myogenin activity: an inactivation mechanism mediated by phosphorylation of myogenin at T87, and a second, novel regulatory mechanism that regulates myg gene activity independently of T87 phosphorylation.

	INTRODUCTION

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Myogenic basic helix-loop-helix (bHLH) proteins, including MyoD, myogenin, Mrf4, and Myf5, bind to E-boxes (CANNTG) in the regulatory regions of a large number of genes that are expressed in skeletal muscle and thereby stimulate their expression (4). These bHLH proteins bind to E-boxes in their own regulatory regions, constituting a feedback network that reinforces their expression once commitment to a myogenic fate has been initiated (5, 29). As such, mechanisms that regulate the expression or activity of myogenic bHLH proteins serve as key regulatory points in muscle differentiation (5, 19).

Multiple posttranslational pathways have been proposed to regulate the activity of myogenic bHLH proteins (21). For example, dimerization with bHLH proteins, such as E12 or E47, leads to the formation of transcriptionally active heterodimers, whereas dimerization with HLH proteins lacking basic regions, such as Id proteins, generates inactive dimers (1). The phosphorylation of myogenic bHLH proteins also regulates their activity, as protein kinase A (PKA) or PKC blocks MyoD and myogenin transcriptional activity (13, 14). Present evidence suggests that PKA-mediated inhibition requires the phosphorylation of proteins other than the myogenic bHLH proteins (13, 31). In contrast, PKC-mediated inhibition requires the phosphorylation of a single threonine residue, T87 in myogenin and T115 in MyoD, in its basic, DNA-binding region. The phosphorylation of this threonine is necessary and sufficient to prevent DNA binding, thereby inhibiting transcription (2, 14).

The PKC-mediated pathway has been proposed to function during development to minimize adventitious myogenic bHLH activity in myoblasts, which might otherwise initiate premature muscle differentiation (14) and, following innervation, to inactivate myogenic bHLH proteins and thereby down-regulate the expression of specific target genes, including acetylcholine receptor (AChR) genes, that depend upon bHLH activity (11, 12). Four lines of evidence support a role for PKC and myogenin in regulating gene expression in innervated muscle. First, myofiber electrical activity suppresses the transcription of specific genes, including AChR subunit genes, in a manner that depends upon the integrity of E-boxes in their regulatory regions (3, 28). Second, pharmacological activation of PKC represses AChR expression in denervated muscle (12). Third, the direct electrical stimulation of denervated muscle increases nuclear PKC activity and down-regulates myg expression (9, 12). Fourth, the level of myogenin phosphorylation is higher in electrically active than in tetrodotoxin-treated, electrically quiescent cultured myotubes (18).

Here, we studied the role of myogenin phosphorylation in vivo. We showed that electrical activity stimulates the phosphorylation of myogenin at T87, but we found that T87 phosphorylation is nonessential for muscle development or for the decrease in gene expression that is associated with an increase in myofiber electrical activity. We observed, however, that the kinetics in the response of the myg gene to innervation is altered both during development and following denervation. Thus, although the phosphorylation of myogenin at T87 has a role in regulating myg expression following innervation, these data indicate that pathways independent of T87 phosphorylation are sufficient to inactivate myg expression during development and in adult muscle.

	MATERIALS AND METHODS

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Surgical procedures. Mice were anesthetized by intraperitoneal injection of ketamine (150 mg/kg of body weight) and xylazine (10 mg/kg). Lower hind limb muscles were denervated by removing an 5-mm section of the sciatic nerve. Denervated gastrocnemius muscles were stimulated in situ for 30 min with a pair of platinum wires placed on top of and below the muscle; the muscles were stimulated with a train of stimuli (100 Hz for 1 s) every 60 s as described elsewhere (16).

Generation of myg^T87N mutant mice. A mouse myg 3.85-kb genomic fragment containing all three exons was a gift of E. Olson. PCR-based mutagenesis was used to convert codon 87, in exon 1, from ACA to AAC. An frt-flanked PGK-neomycin resistance cassette was inserted into the unique BamHI site in intron I, and a ß-actin-diphtheria toxin A cassette was introduced at the 3' end of the genomic fragment to create the targeting construct (Fig. 2A). Surviving electroporated 129/SV embryonic stem (ES) cells were screened by PCR and Southern blotting, which identified two ES cell lines, from 152 clones analyzed, in which the mutant myg gene had recombined with the wild-type locus (Fig. 2B). One cell line, 2B6, was used to generate mice that were heterozygous for the myg^T87Nneo allele. myg^T87Nneo mice were crossed with mice carrying a hACTB::FlpE transgene to remove the frt-flanked neomycin cassette (23).

View larger version (39K): [in this window] [in a new window]   FIG. 2. (A) Generation of mygT87N/T87N mutant mice. The targeting vector contained 3.85 kb of genomic sequence, including all three exons, plus an frt-flanked PGK-neomycin cassette inserted in reverse orientation into intron 1 and a ß actin-diphtheria toxin A (DTA) expression cassette introduced at the 3' end. The probe and PCR primers (arrows) used to detect mygT87Nneo and mygT87N are shown. (B) PCR (data not shown) and Southern blotting identified two ES cell lines (2B6 and 4E3) with homologous recombination events. In wild-type ES cells, the probe hybridizes to a 7.1-kb HincII fragment and a 6.3-kb HincII-SpeI fragment; in targeted ES cells, the probe also hybridizes to a 9.2-kb HincII fragment and a 6.9-kb HincII-SpeI fragment from the targeted locus. Size markers (in kilobase pairs) are shown. (C) mygT87N/mygT87N mice express only mutant mygT87N RNA. RNA from wild-type mice fully protected a myg RNA probe from digestion with RNase (arrowhead), whereas hybridization with RNA from mygT87N/mygT87N mice resulted in two (140- and 190-nucleotide) RNase cleavage products (arrows). Size markers (in nucleotides) are shown. H, HincII; S, SpeI; E, EcoRI; B, BamHI.

Phosphorylation analysis. F5D, a hybridoma cell line that secretes antibodies to myogenin, was kindly provided by W. Wright (32). Antibodies to myogenin phosphorylated at T87 were generated by immunizing rabbits with a phosphopeptide (VDRRRAApTLREK) coupled to keyhole limpet hemocyanin (Research Genetics, Huntsville, Ala.). Limb muscle tissues were homogenized on ice in 20 mM HEPES-150 mM sodium chloride-2.5 mM EDTA (pH 7.4). Following the addition of detergent (0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 1% Triton X-100) and after 20 min on ice, the lysates were precleared by centrifugation. Myogenin was immunoprecipitated from the lysates by overnight incubation with F5D at 4°C. CaMKII and PKC were obtained from Upstate Biotech (Lake Placid, N.Y.) and activated according to the protocols provided by the manufacturers. The plasmid encoding glutathione S-transferase (GST)-myogenin was a gift of E. Olson (6). GST-myogenin was phosphorylated with unlabeled ATP (1 mM; 30 min at 30°C), isolated on glutathione-Sepharose beads, fractionated by SDS-polyacrylamide gel electrophoresis, and electroblotted onto nitrocellulose; the blots were probed with F5D or antibodies to myogenin phosphorylated at T87. Primary antibodies were detected by using horseradish peroxidase-conjugated secondary antibodies to mouse or rabbit immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, Pa.). GST-myogenin was phosphorylated with [-³²P]ATP (0.1 µM, 10 µCi), isolated on glutathione-Sepharose beads, and fractionated by SDS-polyacrylamide gel electrophoresis; the dried gel was exposed to X-ray film for several hours at room temperature.

Histochemistry. NADH reductase activity was visualized by staining frozen sections of skeletal muscle (in 1.5 mM Nitro Blue Tetrazolium-1.5 mM ß-NADH-0.2 M Tris [pH 7.5]) for 30 min at 25°C. Sections were dehydrated and rehydrated through an acetone series before aqueous mounting was carried out. Myosin isoform expression was assessed by staining frozen sections of skeletal muscle with antibodies to type I myosin (A4.840), type II myosin (N3.36), or type IIB myosin (BF-F3). These antibodies were purchased from the American Type Culture Collection (Manassas, Va.) and the Developmental Studies Hybridoma Bank (Iowa City, Iowa).

	RESULTS

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Electrical activity stimulates phosphorylation of myogenin at T87. Myogenin is phosphorylated to a greater extent in spontaneously contracting cultured myotubes than in tetrodotoxin-treated, electrically inactive myotubes (18), but the sites of phosphorylation are not known. We directly stimulated denervated adult muscle and used antibodies directed against a phospho-T87 peptide to compare the levels of myogenin T87 phosphorylation in denervated and denervated-and-stimulated skeletal muscle (Fig. 1A). We stimulated gastrocnemius muscles that had been denervated for 2 days for 30 min in situ and measured T87 phosphorylation in denervated and denervated-and-stimulated muscles by immunoprecipitating myogenin and probing Western blots with antibodies to myogenin phosphorylated at T87. Phosphorylation of T87 increased following electrical stimulation, whereas the level of total myogenin was unchanged (Fig. 1A). These data indicate that electrical activity stimulates the phosphorylation of myogenin at T87 in vivo.

View larger version (48K): [in this window] [in a new window]   FIG. 1. Analysis of myogenin phosphorylation at T87. (A) Two days after denervation, gastrocnemius muscles were electrically stimulated for 30 min. The phosphorylation of myogenin (Myg) at T87 was measured in contralateral denervated (Den) and denervated and stimulated (Den + Stim) muscles by immunoprecipitating Myg with a monoclonal antibody (F5D) and blotting for either Myg phosphorylated at T87 (anti-P-thr87) or Myg (arrow). The level of phospho-T87-Myg was higher in denervated and stimulated muscles than in contralateral denervated control muscles. (B) GST-Myg was phosphorylated by CaMKII or PKC with unlabeled ATP in vitro, and Western blots were probed either with a monoclonal antibody (F5D) to Myg or with antibodies to myogenin phosphorylated at T87. GST-Myg (arrow) was phosphorylated at T87 in vitro by purified PKC and CaMKII. GST-Myg was also phosphorylated by CaMKII or PKC with [-32P]ATP in vitro, and the gels were exposed to X-ray film. IP, immunoprecipitation.

Muscle development appears normal in myg^T87N/T87N mice. To determine the role of T87 phosphorylation in vivo, we replaced the wild-type myg allele with a mutant allele in which asparagine was substituted for T87 (Fig. 2). Because in vitro studies indicate that phosphorylation of T87 inactivates myogenin (14), we reasoned that a failure to phosphorylate T87 might confer resistance to such inactivation, leading to adventitious myogenin activity during myogenesis and elevated myogenin activity in muscle tissue from adult mice. We replaced a T with an N because prior studies revealed that limited substitutions for T87 result in the retention of DNA-binding activity and that T87N myogenin remains a potent transcriptional activator (5, 14).

We generated homozygous mutant mice and found that myg^T87N/T87N mice are healthy and fertile. We stained sections of limb muscle tissues from 7-week-old myg^T87N/T87N mice and found that muscle size, as well as the diameters of individual muscle fibers, was normal (Fig. 3). In addition, immunostaining for myosin heavy-chain isoforms demonstrated normal patterning and percentages of fiber types in the soleus and gastrocnemius muscles (Fig. 3). These data indicate that limb muscle development appears normal in myg^T87N/T87N animals.

View larger version (50K): [in this window] [in a new window]   FIG. 3. Myogenesis appears to be normal in mygT87N/T87N mice. (A) The wet weights of mygT87N/mygT87N hind limb muscles were normal. (B) Limb muscle formation was assessed by staining cross sections of soleus and gastrocnemius muscles with antibodies to myosin heavy chains (MyHCs), which identify slow myofibers and subsets of fast myofibers. Scale bar, 25 µm. (C and D) The numbers and patterns of fast and slow fibers, as well as the diameters of the individual myofibers, appeared to be normal in mygT87N/T87N mice. The mean values and standard deviations from the means are indicated; we analyzed two or three mice for each genotype. TA, tibialis anterior; EDL, extensor digitorum longus.

Postnatal down-regulation of myg transcription is delayed in muscle tissue from myg^T87N/T87N mice. myg expression peaks during late embryogenesis and decreases perinatally, presumably due to innervation (9). We assessed whether phosphorylation of myogenin at T87 is required to down-regulate myg expression in innervated muscle by measuring myg RNA levels in postnatal mice. In wild-type mice, myg RNA levels decreased (ca. threefold) between postnatal days 0 and 12 (Fig. 4A) (9). myg expression also decreased postnatally in sibling myg^T87N/T87N mice but remained elevated (1.5-fold) relative to that of wild-type mice at each time point examined (Fig. 4A). Ultimately, by 7 weeks after birth, myg transcript levels in wild-type and myg^T87N/T87N mice were similar (Fig. 4A). These data indicate that a failure to phosphorylate myogenin at T87 results in a delay in myg down-regulation but that mechanisms independent of T87 phosphorylation ultimately inactivate myg expression in postnatal muscle.

View larger version (41K): [in this window] [in a new window]   FIG. 4. myg and AChR expression in innervated and denervated muscle tissues from mygT87N/T87N mutant mice. myg (A and C) and AChR -subunit (B and D) RNA expression was measured by RNase protection in postnatal muscles from postnatal day 0 (P0) to P49 (A and B) and in limb muscles, from 6-week-old mice, that had been denervated for 8, 16, 24, or 48 h (C and D). myg and AChR -subunit expression levels were indexed to GAPDH expression; the means ± standard errors of the means are indicated. The numbers of samples were 4 (A), 2 (B), and 3 or 4 (C and D) for each time point.

Multiple innervation-dependent pathways inactivate myg expression. Our results indicate that innervation ultimately inactivates myg expression independently of T87 phosphorylation. A failure to phosphorylate myogenin at T87, however, delays the rate at which myg expression is down-regulated postnatally. These findings raised the possibility that a failure to phosphorylate myogenin at T87 might alter the kinetics in the response of myg to denervation. myg expression is low in innervated adult muscle and increases by more than 1 order of magnitude within the first day following denervation. We denervated limb muscles and measured the rate of increase in myg RNA expression shortly after denervation. In wild-type mice, as reported previously (9), myg RNA expression reached peak levels (an 75-fold increase) by 48 h following denervation (data not shown). In wild-type mice, expression increased modestly during the first 16 h after denervation, 1.4-fold at 8 h and 2.3-fold at 16 h (Fig. 4C) (9). In contrast, in myg^T87N/T87N mice, myg expression increased more rapidly, 4.5-fold at 8 h and 6.2-fold at 16 h (Fig. 4C). Ultimately, by 24 h after denervation, myg expression levels in myg^T87N/T87N mice and wild-type siblings were similar (Fig. 4C). At this time and thereafter, AChR expression levels were equally elevated in wild-type and myg^T87N/T87N mice (Fig. 4D). These data suggest that two differing innervation-dependent mechanisms inhibit myogenin: a mechanism that is dependent upon the phosphorylation of T87, and a second mechanism that is independent of phosphorylation of myogenin at T87 and that is lost rapidly after denervation. Our experiments indicate that within several hours after denervation, the latter inactivation mechanism is no longer effective, allowing the latent transcriptional potential of T87N myogenin to be revealed (Fig. 5).

View larger version (52K): [in this window] [in a new window]   FIG. 5. Model for two differing innervation-dependent mechanisms that inhibit myogenin. In innervated (Inn), electrically active muscle, the transient release of calcium from intracellular stores and/or the entry of extracellular calcium through voltage-activated channels increases the activity of PKC and CaMKII, resulting in the phosphorylation of Myg at T87, inhibiting Myg from binding E-boxes and activating myg transcription. Electrical activity stimulates a second inactivation pathway, which may inhibit transcription mediated by proteins that bind a MEF2 site and/or a MEF3 site in the myg regulatory region. This second inactivation pathway is rapidly lost following denervation (Den, 12 h), but myg transcription resumes only after sufficient Myg phosphorylated at T87 (Myg-T87-P) is dephosphorylated and/or adequate Myg is synthesized to activate myg transcription (Den, 24 h). In mygT87N/T87N mutant mice, once the second inactivation mechanism is no longer effective (Den, 12 h), the latent transcriptional potential of T87N myogenin is apparent and results in a premature increase in myg transcription.

	DISCUSSION

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As myogenin can bind to E-boxes in the myg promoter and stimulate transcription (8), the T87 myogenin-independent inactivation mechanism may involve posttranslational modifications of T87N myogenin that inactivate its ability to bind DNA or to stimulate transcription. Alternatively, this inactivation mechanism may involve transcriptional repression of myg, mediated through cis-acting elements other than E-boxes (Fig. 5). In this regard, MEF2, a MADS-box containing a transcription factor essential for muscle development (15), binds to the myg regulatory region and stimulates myg expression (8). Thus, mechanisms that down-regulate MEF2 activity may be sufficient to inhibit myg^T87N/T87N expression (7, 33).

A failure to phosphorylate myogenin at T87 is insufficient to activate myg expression in electrically active, innervated muscle tissue. Likewise, the disruption of the other inactivation pathway(s) may be insufficient to express myg in innervated muscle, as the phosphorylation of T87 may be adequate to ensure that myg expression remains inhibited. An understanding of how the multiple pathways regulate myg expression will require insight into the nature of the other inactivation pathway(s).

	ACKNOWLEDGMENTS

 
We thank E. Olson for providing us with the mouse myogenin gene and for helpful discussions. We thank W. Wright for the antibody to myogenin and S. Clark for his comments on the manuscript.

This work was supported by a research grant from the NIH to S.J.B. (NS27963) and by postdoctoral fellowships from the NIH to L.F. and from the HFSP to C.S.B.

	FOOTNOTES

 
* Corresponding author. Mailing address: Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, New York University Medical School, 540 First Ave., New York, NY 10016. Phone: (212) 263-7341. Fax: (212) 263-2842. E-mail: burden{at}saturn.med.nyu.edu.

Present address: Center for Medical Education, Ball State University, Muncie, IN 47303-4609.

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