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Originally published In Press as doi:10.1074/jbc.M608265200 on February 26, 2007

J. Biol. Chem., Vol. 282, Issue 16, 11765-11775, April 20, 2007
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Regulation of a Transcript Encoding the Proline-rich Membrane Anchor of Globular Muscle Acetylcholinesterase

THE SUPPRESSIVE ROLES OF MYOGENESIS AND INNERVATING NERVES*

Heidi Q. Xie, Roy C. Y. Choi, K. Wing Leung, Nina L. Siow1, Ling W. Kong, Faye T. C. Lau, H. Benjamin Peng, and Karl W. K. Tsim2

From the Department of Biology and the Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China

Received for publication, August 29, 2006 , and in revised form, February 23, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcriptional regulation of proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G4 AChE), was revealed in muscle during myogenic differentiation under the influence of innervation. During myotube formation of C2C12 cells, the expression of AChET protein and the enzymatic activity were dramatically increased, but the level of G4 AChE was relatively decreased. This G4 AChE in C2C12 cells was specifically recognized by anti-PRiMA antibody, suggesting the association of this enzyme with PRiMA. Reverse transcription-PCR analysis revealed that the level of PRiMA mRNA was reduced during the myogenic differentiation of C2C12 cells. Overexpression of PRiMA in C2C12 myotubes significantly increased the production of G4 AChE. The oligomerization of G4 AChE, however, did not require the intracellular cytoplasmic tail of PRiMA. After overexpressing the muscle regulatory factors, myogenin and MyoD, the expressions of PRiMA and G4 AChE in cultured myotubes were markedly reduced. In addition, calcitonin gene-related peptide, a known motor neuron-derived factor, and muscular activity were able to suppress PRiMA expression in muscle; the suppression was mediated by the phosphorylation of a cAMP-responsive element-binding protein. In accordance with the in vitro results, sciatic nerve denervation transiently increased the expression of PRiMA mRNA and decreased the phosphorylation of cAMP-responsive element-binding protein as well as its activator calcium/calmodulin-dependent protein kinase II in muscles. Our results suggest that the expression of PRiMA, as well as PRiMA-associated G4 AChE, in muscle is suppressed by muscle regulatory factors, muscular activity, and nerve-derived trophic factor(s).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
During cholinergic transmission at neuron-to-neuron synapses in the central nervous system or neuromuscular junctions (nmjs)3 in the peripheral nervous system, acetylcholinesterase (AChE; EC 3.1.1.7 [EC] ) plays a crucial role in terminating the synaptic transmission by hydrolyzing the neurotransmitter acetylcholine. Depending on alternative splicing in the 3' region of the primary transcript, AChE exists in different molecular forms (1). This process generates different subunits that contain the same catalytic domain but with distinct carboxyl termini (1, 2). In mammals, the AChER variant produces a soluble monomer that is up-regulated in the brain during stress (3); the AChEH variant produces a glycosylphosphatidylinositol-anchored dimer that is mainly expressed in blood cells; the AChET variant is the only subunit expressed in the brain and muscle. AChET subunits form nonamphiphilic tetramers with a collagen tail (ColQ) as asymmetric AChE (A12 AChE) in muscle and also form amphiphilic tetramers associated with a proline-rich membrane anchor (PRiMA) as globular form AChE (G4 AChE) in brain and muscle (4, 5).

G4 AChE has been found in mammalian tissues, including brain, muscle, and heart (2); its expression pattern exhibits a close resemblance to PRiMA RNA expression (6). In addition to the key role of AChE in cholinergic function, the correct orientation of AChE catalytic subunits at the cell surfaces of certain neurons, targeted by PRiMA, is proposed to be required for neurite outgrowth (7). Additionally, G4 AChE in the brain is related to amyloid plaques and neurofibrillary tangles in Alzheimer disease and may contribute to its development (8). Thus, G4 AChE may have distinct functions in different tissues.

Although G4 is not the major form of AChE in muscle, its existence is tightly controlled. Several studies have revealed that the level of G4 AChE is controlled by the dynamic activity of skeletal muscles. In mammals, fast twitch muscles contain a high amount of G4, whereas slow twitch muscles contain a much smaller amount (9). Alteration of the G4 AChE level after muscle denervation strongly suggests a critical role of motor nerves in G4 AChE regulation (2, 10). The motor nerves may achieve this regulation by two distinct mechanisms: release of trophic factor and nerve-evoked electrical activity. Among the known nerve-derived trophic factors, calcitonin gene-related peptide (CGRP), a neuropeptide with 37 amino acids, which has been identified in spinal cord motor neurons (11), exerts an innervation-like effect to suppress G4 AChE when applied in muscles (12). In addition, CGRP regulates the synthesis of the AChET subunit (13–15) and of acetylcholine receptors (11). On the other hand, exercise induces a marked change in the level of this enzyme form, without modification of other molecular species (16–18). Unfortunately, G4 AChE was analyzed only by sedimentation, and the expression of PRiMA, the only G4-specific component, has not been studied under physiological conditions.

In this study, we sought to identify PRiMA-associated G4 AChE in cultured C2C12 muscle cells and to analyze the expression of mRNAs encoding PRiMA, as well as AChET, in cultured C2C12 cells during myogenic differentiation, the influence of nerve-derived factors and muscular activity, and the effect of denervation in fast twitch and slow twitch muscles. Our results indicate that myogenic regulatory factors (MRFs), muscular activity, and CGRP suppress the expression of PRiMA, probably mainly by activating the cAMP-responsive element-binding protein (CREB) transcription factor. In addition, the production of G4 AChE in muscle is shown to be controlled by the level of PRiMA expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures—The mouse C2C12 muscle cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Undifferentiated C2C12 myoblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum and incubated at 37 °C in a water-saturated 5% CO2 incubator. All reagents for cell cultures were from Invitrogen. Myogenic differentiation was induced as previously described (19). In brief, the cultured myoblasts were allowed to grow in Dulbecco's modified Eagle's medium with 10% fetal bovine serum until they were confluent, and then they were changed to Dulbecco's modified Eagle's medium with 2% heat-inactivated horse serum to induce differentiation. In the myogenesis studies, cell lysates were collected on each day starting from the first day of induction (day 0) to the eighth day (day 7), and the extracts were stored at –80 °C. The drug treatments were carried out on 4-day-old myotube cultures. The human embryonic kidney (HEK) 293T fibroblast cell line was obtained from the ATCC and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a water-saturated 5% CO2 incubator.

DNA Construction and Transfection—cDNAs encoding full-length mouse PRiMA (PRiMA I), and a COOH-terminal truncated mutant (PRiMA I{Delta}C-term; obtained by deleting the COOH-terminal region, residues 122–153) were tagged by FLAG epitope (obtained by inserting a FLAG epitope of DYKDE at position 36 between the putative signal sequence and the NH2 terminus) in pER-BOS mammalian expression vector (6). The mouse myogenin and MyoD cDNAs were described by Lee et al. (20). Vectors expressing the CREB (wild-type) and K-CREB (inactive mutant) cDNAs were purchased from Clontech (Mountain View, CA). The cDNA encoding the constitutively active form of rat calcium/calmodulin-dependent protein kinase II-{gamma} (CaMKII-{gamma}) was subcloned into pCS2MT vector (21). Transient transfection of myoblasts with the cDNA construct was performed with a Lipofectamin Plus reagent (Invitrogen), according to the manufacturer's instructions. The transfection efficiency was consistently 30–40% in the C2C12 myoblasts. The transfection in the cultured HEK293T fibroblasts was done by calcium phosphate precipitation as described previously (15).

Production and Purification of Anti-PRiMA Polyclonal Antibody—The mouse PRiMA (from amino acid 114 to 153)-glutathione S-transferase fusion protein was expressed in BL21 (DE3) pLysE Escherichia coli (Invitrogen) and purified by glutathione bead chromatography (Amersham Biosciences) according to the manufacturer's instructions. After digestion by thrombin (Sigma), the PRiMA-(114–153) antigen was purified by Superdex 75 10/300 gel filtration chromatography (Amersham Biosciences). Polyclonal antibodies were raised in a 2-kg male New Zealand White rabbit by immunization with 750 µg of antigen, mixed with an equal volume of complete Freund's adjuvant (Sigma). The immunization was carried out with the same amount of antigen three times within 1 month. The anti-PRiMA serum was collected and purified by protein G-Sepharose (Amersham Biosciences) according to the manufacturer's instructions. The amount of purified antibody was determined spectrophotometrically.

Drug Treatments—Four-day-old cultured myotubes were treated with either acetylcholine chloride (ACh; 10 and 100 µM), depolarizing agent potassium chloride (KCl; 10 or 20 mM), Ca2+ ionophore A23187 [GenBank] (0.2 or 0.5 µM), CGRP (1 µM), or N6, O2'-dibutyryl-cAMP (Bt2-cAMP; 0.3 and 1 mM) for 2 days. Pretreatment with KN62 (20 µM; an inhibitor of CaMKII) was done for 3 h before the drug application. In phosphorylation analyses, myotube cultures were serum-starved for 3 h before the drug application. All of the drugs were purchased from Sigma.

Sciatic Nerve Denervation—Two-month-old Sprague-Dawley rats weighing ~250 g were anesthetized by isoflurane. Denervation was performed by removing a ~3-mm portion of the sciatic nerve located around the upper thigh by an aseptic surgical technique (22). Rats were sacrificed according to the instructions of the Animal Care Facility at Hong Kong University of Science and Technology. Soleus and tibialis muscles were collected on days 1, 2, 5, and 8 after denervation. Muscle samples were frozen in liquid nitrogen immediately after dissection and stored at –80 °C before the RNA or protein extraction. Control experiments were performed by sham operations on the same muscles of different rats.

Real Time Quantitative PCR—Total RNA from either C2C12 cultures or rat tissues was isolated by TRIzol reagent (Invitrogen), and 5 µg of RNA was reverse-transcribed by Moloney murine leukemia virus reverse transcriptase (Invitrogen), according to the manufacturer's instructions. Real time PCR of PRiMA, AChET, and glyceraldehyde-3-phosphate dehydrogenase transcripts was performed on equal amounts of reverse-transcribed products, using SYBR Green Master mix and Rox reference dye, according to the manufacturer's instructions (Applied Bioscience, Foster City, CA). The primers were as follows: 5'-TCT GAC TGT CCT GGT CAT CAT TTG CTA C-3' and 5'-TCA CAC CAC CGC AGC GTT CAC-3' for mouse PRiMA I and II (GenBankTM numbers NM 133364 and NM 178023); 5'-CTG GGG TGC GGA TCG GTG TAC CCC-3' and 5'-TCA CAG GTC TGA GCA GCG TTC CTG-3' for mouse AChET (23); and 5'-AAC GGA TTT GGC CGT ATT GG-3' and 5'-CTT CCC GTT CAG CTC TGG G-3' for mouse and rat glyceraldehyde-3-phosphate dehydrogenase (21). The SYBR green signal was detected by a Mx3000pTM multiplex quantitative PCR machine (Stratagene, La Jolla, CA). The transcript expression levels were quantified by using the Ct value method (24), where values were normalized to glyceraldehyde-3-phosphate dehydrogenase as an internal control in the same sample. The PCR products were analyzed by gel electrophoresis, and the specificity of amplification was confirmed by the melting curves.

Immunochemical Analysis—C2C12 cultures, cDNA-transfected HEK 293T cultures, and muscle and brain tissues were homogenized in a lysis buffer (10 mM HEPES, pH 7.5, 1 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, and 1 mg/ml bacitracin), followed by centrifugation at 12,000 x g for 20 min at 4 °C. Protein samples were denatured at 100 °C for 5 min in a buffer containing 1% SDS and 1% dithiothreitol and separated by 8 or 12% SDS-polyacrylamide gel electrophoresis. In the Western blot analysis, we used anti-PRiMA polyclonal antibody (purified at 0.5 µg/ml), anti-AChET antibody (1:5,000; BD Biosciences), anti-FLAG antibody (1:1,000; Sigma), anti-myogenin, and anti-MyoD antibodies (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-{alpha}-tubulin antibody (1:5,000; Sigma), anti-phospho-CaMKII and anti-total CaMKII antibodies (1:1,000; Upstate, Billerica, MA), and anti-phospho-CREB and anti-total CREB antibodies (1:1,000; Cell Signaling Technology, Danvers, MA). The immune complexes were visualized using the ECL method (Amersham Biosciences). The intensities of the bands in the control and stimulated samples, run on the same gel and under strictly standardized ECL conditions, were compared on an image analyzer, using, in each case, a calibration plot constructed from a parallel gel with serial dilutions of one of the samples. In the immunofluorescent analysis, the cDNA-transfected HEK293T cells, after 2 days of transfection, were fixed by 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline for 5 min, followed by 50 mM ammonium chloride (NH4Cl) treatment for 25 min. Cultures were permeabilized and blocked by 5% fetal bovine serum, 0.1% Triton X-100 in phosphate-buffered saline for 1 h at room temperature. Anti-PRiMA antibody (2 µg/ml) and anti-FLAG antibody (dilution 1:500) were applied to the cells for 16 h at 4 °C followed by the corresponding Alexa 488-conjugated anti-rabbit secondary antibody for 2 h at room temperature. The cells were dehydrated serially with 50, 75, 95, and 100% ethanol and mounted with a fluorescence mounting medium (DAKO, Carpinteria, CA). The samples were then examined by a Leica confocal microscope with excitation 488 nm/emission 505–550 nm for green color.

Sucrose Density Gradients—Separation of the various molecular forms of AChE was performed by sucrose density gradient analysis, as described previously (15). In brief, sucrose gradients (5 and 20%) in a lysis buffer (10 mM HEPES, pH 7.5, 1 M NaCl, 1 mM EDTA, 1 mM EGTA, and 0.5% Triton X-100) were prepared in 12-ml polyallomer ultracentrifugation tubes with a 0.4-ml cushion of 60% sucrose on the bottom. Cell extracts (0.2 ml) mixed with sedimentation markers (alkaline phosphatase, 6.1 S; beta-galactosidase, 16 S) were loaded onto the gradients and centrifuged at 38,000 rpm in a Sorvall TH 641 rotor at 4 °C for 16 h. Approximately 45 fractions were collected, and the AChE enzymatic activity was determined according to the method described by Ellman (25) with the modification of adding 0.1 mM tetraisopropylpyrophosphoramide, an inhibitor of butyrylcholinesterase, to each fraction. The absorbance at 410 nm was recorded as a function of the reaction time. The amount of the various AChE forms was determined by summation of the enzymatic activities corresponding to the peaks of the sedimentation profile. In the immunoprecipitation of G4 AChE by anti-PRiMA antibody, brain, muscle, and C2C12 cell extracts (1 ml in 10 mM HEPES, pH 7.5, 1 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, and 1 mg/ml bacitracin) were incubated for 4 h at 4 °C with purified anti-PRiMA antibody (10 µg/ml). Then 50 µl of washed protein G-agarose gel (Santa Cruz Biotechnology) was added and incubated for 1 h at 4 °C. After centrifugation, the supernatants were loaded on sucrose gradients for sedimentation analysis.

Other Assays—Protein concentrations were measured routinely using Bradford's method (26) with a kit from Bio-Rad. Statistical tests were run on the PRIMER program, version 1 (40); differences from basal or control values (as shown in the plots) were classified as significant for p < 0.05 (*) and highly significant for p < 0.01 (**) and p < 0.001 (***).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of G4 AChE and PRiMA during Myogenic Differentiation—Cultured mouse C2C12 cells were used as a model system for determining the expression profile of AChE during myogenic differentiation. In the absence of serum, C2C12 myoblasts were allowed to undergo fusion, forming multinucleated myotubes. The Western blots showed that AChET protein (~68 kDa) increased by ~8.5-fold in C2C12 cells during myogenesis, whereas the loading control, {alpha}-tubulin (~55 kDa), remained unchanged (Fig. 1A). In line with the protein profile, the enzymatic activity of AChE dramatically increased (~12-fold) from the myoblast to the myotube stage (Fig. 1A), in agreement with previous results (19).

Sucrose density gradient analysis was used to investigate the AChE molecular forms found during the process of muscle differentiation. At the myoblast stage (day 0), AChE existed predominantly in the G4 form, together with trace amounts of the G1 form (Fig. 1B). When the myoblasts fused to form myotubes on day 4, the relative amount of G4 AChE was reduced, and G1 AChE became the predominant form. A small amount of A12 AChE (ColQ-associated) appeared in mature myotubes on day 7 (Fig. 1B). By quantifying the absolute amount of G1/G4 AChE in muscle during differentiation, we found that the amount of G4 increased by ~4-fold on day 4 (as well as on day 7) of myotube formation; however, the increase in G1 was more robust (over 100-fold) (Fig. 1C). These results reveal that although AChET protein and enzymatic activity are up-regulated during the myogenic differentiation process, the relative proportion of G4 AChE decreases.


Figure 1
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  		FIGURE 1.
Regulation of G4 AChE during C2C12 myogenic differentiation. A, mouse C2C12 myoblasts were induced to differentiate for 7 days by serum depletion. Twenty µg of protein was loaded per lane for Western blots (top). The lower panel shows quantitation of AChET protein from the blots by calibrated densitometry. AChE enzymatic activity was determined by an Ellman assay (25). The data are normalized and expressed as the ratio to the values obtained at day 0 (myoblast stage) arbitrarily set to 1. B, C2C12 cultures were collected to analyze different AChE molecular forms in sucrose density gradients. Equal amounts of AChE activity in 0.2 ml were loaded per sample. AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and one representative result is shown, n = 4. C, the absolute amounts of G1 and G4 AChE were determined from the values obtained in A and B. The data are normalized and expressed as the ratio to the values obtained at day 0 (myoblast stage) of a G4 amount that is arbitrarily set to 1. Values are means ± S.E., n = 4, each with triplicate samples.

 


Figure 2
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  		FIGURE 2.
Identification of PRiMA-associated AChE in C2C12 cells by using anti-PRiMA antibody. A, FLAG-tagged PRiMA cDNA (different amounts) was transfected into cultured HEK293T cells for 2 days. Twenty µg of protein from the collected cell lysate was loaded per lane for Western blots. Both anti-PRiMA and anti-FLAG antibodies were used to recognize the PRiMA band. In the blocking experiment, excess amounts of recombinant PRiMA peptide (from residue 114 to 153) at 5 µg/ml were incubated with anti-PRiMA antibody (0.5 µg/ml) for 4 h at 4 °C before it was applied to the Western blotting. B, the DNA-transfected HEK293T cells as in A were stained with anti-PRiMA and anti-FLAG antibodies as described under "Experimental Procedures." The control was stained with preimmune serum. C, 30 µgof protein from extracts of C2C12 cells (myoblast or myotube), adult rat tibialis, and brain was loaded per lane for Western blots. The blocking by PRiMA peptide was done as in A. D, 1 ml of extract from adult rat brain, muscle (tibialis), or cultured C2C12 myoblasts was incubated with anti-PRiMA antibody (Ab) (10 µg/ml). After the precipitation by protein G-agarose, the supernatant (0.2 ml) was subjected to sucrose density gradient analysis. Preimmune serum was used in the control. AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and one representative result is shown, n = 3. Bar, 10 µm.

 
Besides in brain enzymes, the association of PRiMA with G4 AChE has not been identified biochemically in other tissues. We generated anti-PRiMA polyclonal antibody to address this gap. In FLAG-tagged PRiMA cDNA-transfected HEK293T cells, both anti-PRiMA and anti-FLAG antibodies recognized a protein band of ~20 kDa, corresponding to the predicted size of PRiMA, in our Western blots (Fig. 2A, left). The recognition could be blocked by the preincubation of anti-PRiMA antibody with excess amounts of PRiMA peptides. This did not occur with anti-FLAG antibody (Fig. 2A, right). In parallel, the antibody recognition was further confirmed in the immunofluorescent staining of FLAG-tagged PRiMA cDNA-transfected cells (Fig. 2B). These results indicated the specificity of our anti-PRiMA antibody.

By using the anti-PRiMA antibody in the Western blots, a band of ~20 kDa was recognized in the extracts derived from C2C12 myoblasts and myotubes; the expression level was relatively higher in myoblasts (Fig. 2C, left). In addition, PRiMA was also detected in muscle (tibialis). Serving as a control, the brain extract showed similar band recognition. Again, the antibody recognition was blocked by PRiMA peptides in all cases (Fig. 2C, right). To understand the association of PRiMA with G4 AChE, the brain, muscle, and C2C12 cell extracts were immunoprecipitated by anti-PRiMA antibody. As shown in Fig. 2D, the G4 AChE in the extracts was depleted by the antibody treatment with ~70% depletion in brain, ~40% depletion in muscle, and ~40% depletion in cultured C2C12 cells. Clearly, the depletion was more robust in the brain extracts. The G1 enzyme in both cases was not affected by this antibody. These results suggest that a major part of G4 AChE in muscle is associated with PRiMA. The identity of the rest of G4 AChE was not further analyzed.

A reduction in the expression of PRiMA may explain the reduction in G4 AChE during the myogenic differentiation process. According to Perrier et al. (27), two splicing variants of PRiMA mRNAs are generated from the PRiMA gene to produce different proteins (PRiMA I and PRiMA II; Fig. 3A). PRiMA I mRNA possesses exons 4 and 5 and produces a 40-residue-long intracellular cytoplasmic tail, whereas PRiMA II mRNA possesses exons 4, 4b, and 5, resulting in a short intracellular motif (Fig. 3A). To differentiate these two PRiMA isoforms, reverse transcription-PCR was performed by specific primers located in exon 4 and exon 5. In C2C12 cultures, a large amount of PRiMA I was found, whereas PRiMA II was barely detectable (Fig. 3B). Similarly, both the tibialis (fast) and soleus (slow) muscles predominantly expressed PRiMA I (Fig. 3B). In adult rat brain, both isoforms of PRiMA exist, which means that rat brain can serve as a positive control, as reported previously (27).


Figure 3
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  		FIGURE 3.
Reduction of PRiMA mRNA expression during myotube formation. A, genomic structure of the PRiMA gene shows the alternative splicing in forming PRiMA I and II. PRiMA II contains an additional exon, 4b, resulting in a short NQAI motif. B, total RNAs were extracted from cultures or tissues to perform reverse transcription-PCR analysis to determine the presence of PRiMA I (~145 bp) and II (~302 bp). Rat brain has both mRNAs. Glyceraldehyde-3-phosphate dehydrogenase mRNA (~657 bp) served as controls in showing the equal loading of RNA. One of the representative results is shown, n = 3. C, quantitative real time PCR was performed to reveal the mRNA expressions of AChET and PRiMA I and II (as in B) in C2C12 during differentiation. Data are normalized and expressed as the percentage of the control (day 0).

 
Regulations in the level of PRiMA mRNA were determined by quantitative real time PCR analysis using the same sets of primers as in Fig. 3B. This showed that PRiMA I was expressed at high levels in the myoblast stage; the expression declined after the onset of differentiation; and finally it reached a low level in the myotube stage (Fig. 3C). The level of PRiMA mRNA was reduced by at least 50% during myogenesis. In contrast, the level of AChET mRNA increased dramatically (~120-fold) from the myoblast stage to the myotube stage, in agreement with the up-regulation of AChET protein and the enzymatic activity. On the other hand, PRiMA II mRNA was maintained at a low level throughout the entire differentiation process; its expression was not further investigated. Therefore, we refer to PRiMA I as PRiMA hereafter, unless otherwise specified.

The down-regulation of PRiMA mRNA in C2C12 cultures during myogenesis is consistent with the observed reduction of G4 AChE. In order to determine the possible role of PRiMA in directing the formation of G4 AChE in muscle, C2C12 myoblasts were co-expressed with cDNAs encoding AChET and PRiMA I; the molecular forms of AChE were analyzed subsequently at the myotube stage. Overexpression of AChET in the cultures produced mostly G1 AChE, with minor amounts of G4 and A12 AChE (Fig. 4A), in agreement with the endogenous expression of AChE in the myotubes. Overexpression of PRiMA together with AChET markedly increased G4 AChE in the myotubes (Fig. 4B), indicating that the oligomerization of G4 AChE was directed by PRiMA. Overexpression of PRiMA alone did not significantly increase the amount of G4 AChE in the cultures; this might be due to the limited supply of AChET for G4 AChE assembly.

To determine the possible requirement of the cytoplasmic tail of PRiMA I in G4 AChE oligomerization, this tail region was deleted to form PRiMA I{Delta}C-term (Fig. 4C). This truncated cDNA construct, resembling PRiMA II, was co-expressed with AChET cDNA in C2C12 cultures. The truncated PRiMA I{Delta}C-term markedly increased the production of G4 AChE (Fig. 4C) in the same manner as the full-length PRiMA I. These results clearly indicate that PRiMA I and PRiMA II are able to direct the assembly of AChET into G4 AChE in muscle, and the intracellular cytoplasmic tail of PRiMA I is not required in this oligomerization process. Thus, the decrease of G4 AChE during myogenic differentiation could be mainly attributed to the down-regulation of PRiMA.

Myogenic Regulatory Factors Regulate PRiMA Expression—In order to elucidate the mechanism that suppresses the transcription of the PRiMA gene during muscle differentiation, we investigated the possible role of MRFs. Among different muscle-specific transcription factors, myogenin (28) and MyoD (29) have been shown to play roles in the early phase of myotube formation. During C2C12 differentiation, myogenin (~36 kDa) and MyoD (~38 kDa) were found to be induced, reaching a maximal expression at the onset of the differentiation process and then declining after 4 days of fusion and subsequently remaining at a low level in mature myotubes (Fig. 5A).

To determine the regulatory role of MRFs on PRiMA mRNA expression, C2C12 myoblasts were transfected with cDNAs encoding myogenin and MyoD and allowed to form myotubes. Overexpression of myogenin and MyoD decreased the expression of PRiMA mRNA to ~50%, as compared with the mock control pcDNA3 (Fig. 5B). In contrast, the level of AChET mRNA increased by overexpressing myogenin and MyoD in muscle cultures; the increase of AChET protein was correlated with an increase of enzymatic activity by ~60%. These observations are in line with the notion that MRFs suppress PRiMA expression during the early stages of myogenesis.

We also analyzed the molecular forms of AChE in myogenin or MyoD cDNA-transfected myotubes. The pcDNA3-transfected myotubes expressed mostly G1 and G4 AChE with a minor portion of A12 AChE (Fig. 5C). When myogenin or MyoD was overexpressed in the myotubes, the total amount of G4 AChE within the transfected myotubes was reduced by over 50%. In contrast, the amount of A12 AChE was increased by ~50% under the effects of the MRF overexpression (Fig. 5, C and D). The reduction of G4 AChE was probably due to the decrease of PRiMA expression under the control of the overexpressed MRFs.

Muscular Activity and CGRP Suppress PRiMA Expression—In vertebrate nmjs, the innervated motor axon provides two types of anterograde signals, ACh-induced muscular activity and nerve-derived factors, to control the expression of postsynaptic genes in muscle. Muscular activity is known to suppress acetylcholine receptor expression via intracellular Ca2+ and CaMKII (22, 30). To mimic this muscular activity in myotube cultures, ACh (10 and 100 µM), depolarizing agent KCl (10 and 20 mM), and Ca2+ ionophore A23187 [GenBank] (0.2 and 0.5 µM) were applied to the myotube cultures for 2 days, and then the expressions of PRiMA and AChET mRNAs were determined. Compared with the controls, all of the drug treatments reduced the expression of PRiMA mRNA (Fig. 6A). This down-regulation effect, induced by muscular activity, was also observed in the gene transcription of AChET. These results suggest that nerve-evoked muscular activity provides a uniform signal across the entire muscle fiber to reduce the expressions of PRiMA and AChET.


Figure 4
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  		FIGURE 4.
Induction of G4 AChE by overexpression of PRiMA. In C2C12 myotubes, AChET was overexpressed together with the control vector (A), with PRiMA I (B), or with PRiMA I{Delta}c-term (C). The transfected C2C12 cultures were collected to analyze different AChE molecular forms by sucrose density gradient analysis. Equal amounts of AChE activity in 0.2 ml were loaded per sample. AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and one representative result is shown, n = 3.

 
CGRP, a nerve-derived trophic factor, is able to trigger an accumulation of intracellular cAMP, which has been demonstrated to regulate the synthesis and formation of G4 AChE in muscles (13–14, 31). Total RNAs were extracted from CGRP (1 µM)-treated or Bt2-cAMP (1 and 3 mM)-treated cultures for quantitative real time PCR analysis. The amount of PRiMA mRNA was reduced to ~30% by CGRP or Bt2-cAMP (Fig. 6A). Expression of AChET mRNA was also reduced to ~50% in the drug-treated samples, as reported previously (19). In parallel with the muscular activity- and CGRP-induced PRiMA down-regulation, the amount of G4 AChE in cultured myotubes was selectively decreased by these treatments; the reduction of G4 AChE was over 40% (Fig. 6, B and C). In contrast, the expression of the total A12 AChE remained almost unchanged.

CREB Phosphorylation Mediates PRiMA Suppression—The phosphorylation of CREB is one of the downstream signals in CGRP/cAMP-induced signaling cascades and has been demonstrated to be a key regulator in suppressing the expression of AChET in muscle (15). Here, we determined its possible role in PRiMA suppression. In serum-starved myotubes, application of CGRP at 1 µM induced the phosphorylation of CREB (~43 kDa), which was recognized by an anti-phospho-CREB antibody (Fig. 7A). We observed a transient CGRP-induced CREB phosphorylation, with a peak of activation (~6-fold) 10 min after CGRP challenge. The total amount of CREB at ~43 kDa remained unchanged at different time intervals. Similarly, application of muscular activity-inducing agents (ACh and A23187 [GenBank] ) also induced CREB phosphorylation; the phosphorylation was sustained for a longer time with these agents, and the peak occurred 10–15 min after the drug challenge (Fig. 7, A and B). Although ACh should be rapidly hydrolyzed, its effect on CREB phosphorylation peaked at 10 min after the treatment. This result agrees with the role that ACh plays in affecting the formation of AChE forms.


Figure 5
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  		FIGURE 5.
Suppression of PRiMA expression by myogenic transcription factors. A, expression profiles of myogenic regulatory factors, myogenin (~36 kDa) and MyoD (~38 kDa), were revealed during C2C12 differentiation. Twenty µg of protein was loaded per lane for Western blotting. The lower panel shows quantitation of the protein from the blots by calibrated densitometry. Data are normalized and expressed as the ratio to basal reading, where day 0 (basal) equals 1. B, myogenin or MyoD was overexpressed in C2C12 myotubes. Total RNAs or cell lysates were extracted from the cultures. Quantitative real time PCR was performed to reveal the mRNA expressions of AChET and PRiMA I. AChE activity was determined by an Ellman assay (25). Data are normalized and expressed as the percentage of the control (pcDNA3-transfected cultures). C, cell lysates obtained from B were subjected to sucrose density gradient analysis. An equal amount of AChE activity in 0.2 ml was loaded per sample. AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and one representative result is shown, n = 3. D, the absolute amounts of different AChE forms were quantified from B and C. The data are normalized and expressed as a ratio to the values obtained at control culture of G4 amount that is arbitrarily set to 1. All values are mean ± S.E., n = 4, each with triplicate samples. In this and other figures, the difference from the control or basal level (except where noted) is as follows: *, significant (p < 0.05); **, highly significant (p < 0.01); ***, highly significant (p < 0.001).

 
These results are consistent with the fact that CGRP induced CREB phosphorylation. The CGRP-induced CREB phosphorylation is mediated by a cAMP-dependent signaling pathway, via a CGRP receptor complex on the muscle surface (15, 32). In contrast, the mechanism of muscular activity-induced CREB phosphorylation has not yet been determined. CaMKII is one of the downstream effectors of the muscular activity-inducing agents (22, 30) and therefore could be responsible for the CREB phosphorylation. In cultured myotubes, application of activity-inducing agents (ACh and A23187 [GenBank] ) induced a sustained phosphorylation of CaMKII (~50 kDa) that was at least 5-fold stronger (Fig. 7, A and B). The total amount of CaMKII remained unchanged at different time intervals. Additionally, the activity-induced CREB phosphorylation could be fully blocked by the pretreatment with KN62, a CaMKII inhibitor, in cultured myotubes (Fig. 7C). To confirm the role of CaMKII in phosphorylating CREB, we overexpressed an active form of CaMKII in cultured myotubes; this overexpression induced an activation of CREB (Fig. 7D). These results, therefore, suggest that CREB may act as one of the downstream effectors for CaMKII activation in muscle.

In cultured myotubes, overexpression of CREB decreased AChET and PRiMA mRNAs in a dose-dependent manner; the reduction was more significant for PRiMA mRNA (Fig. 8A) than for AChET. The role of CREB in directing the suppression of PRiMA expression by CGRP and muscular activity-inducing agents was further demonstrated by using a dominant negative mutant of CREB (K-CREB). Overexpression of K-CREB in cultured C2C12 myotubes markedly reduced the decrease of PRiMA induced by ACh, KCl, CGRP, and Bt2-cAMP (Fig. 8B). These results support the argument that CREB plays a key role in the regulation of PRiMA expression in muscle.

The suppression of PRiMA expression by CREB could be further confirmed by comparing the expression of PRiMA mRNA in fast twitch (tibialis) and slow twitch (soleus) muscles. The basal levels of PRiMA and AChET mRNAs in tibialis were ~10- and ~6-fold higher, respectively, than those in soleus (Fig. 9A). To determine the regulatory effects of innervation on PRiMA expression in muscles, a portion of the sciatic nerve was removed from the rats by surgical denervation. After the denervation, both tibialis and soleus muscles were collected at different time points to examine the PRiMA mRNA expression. Fig. 9B (left) shows an increase of PRiMA mRNA by ~5-fold in soleus and ~2-fold in tibialis, 1 day after the denervation. The levels of AChET mRNA decreased in both types of muscles, starting from 1 day after denervation, which served as a control (Fig. 9B, right). These results strongly suggest that the motor neuron exerts a suppressive effect on PRiMA gene expression in muscle, and this suppression effect could be particularly significant in slow muscles.


Figure 6
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  		FIGURE 6.
Muscular activity and CGRP suppress PRiMA mRNA expression. A, ACh, KCl, A23187, CGRP, and Bt2-cAMP were applied onto cultured myotubes for 2 days. Total RNAs (5 µg) were extracted from the cultures, and quantitative real time PCR was performed to reveal the mRNA expressions of AChET and PRiMA I. Data are normalized and expressed as the percentage of the control (untreated cultures). B, cell lysates obtained from A were subjected to sucrose density gradient analysis. Equal amounts of AChE activity in 0.2 ml were loaded per sample. AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and one representative result is shown, n = 3. C, the absolute amounts of different AChE forms were quantified from B. The data are normalized and expressed as the ratio to the values obtained at control culture of G4 amount that is arbitrarily set to 1. All values are mean ± S.E., n = 4, each with triplicate samples.

 


Figure 7
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  		FIGURE 7.
Muscular activity and CGRP induce the phosphorylation of CREB and CaMKII. A, ACh (100 µM), A23187 (0.5 µM), and CGRP (1 µM) were applied onto cultured myotubes. Twenty µg of protein was loaded per lane in the Western blot for probing total and phosphorylated forms of CREB at ~43 kDa (top) and CaMKII at ~50 kDa (bottom). B, the amount of phosphorylation from the blots in A was quantified by calibrated densitometry. Data are normalized and expressed as the ratio to basal reading, where time 0 (untreated as basal) equals 1. C, KN62 (20 µM) were applied 3 h before adding A23187 (0.5 µM) onto cultured myotubes for 15 min. The detection of CREB and its phosphorylated form was as in A. D, the cDNA encoding rat CaMKII was overexpressed in C2C12 myotubes for 2 days. Different amounts of the DNA were used. The detection of total CREB and its phosphorylated form was as in A. All values are mean ± S.E., n = 4.

 
To correlate this nerve-induced suppressive effect with CaMKII and CREB phosphorylation, the levels of active phosphorylated forms of CaMKII and CREB were determined in fast and slow muscles. Both tibialis and soleus expressed similar amounts of phosphorylated forms of CaMKII. In contrast, the soleus contained higher levels (over 5-fold higher) of active forms of CREB than the tibialis (Fig. 9C). One day after denervation, both muscles showed a reduction of CaMKII and CREB phosphorylation by at least 40% as compared with the sham-operated control muscles (Fig. 9, C and D); this reduction appeared early in soleus, and it was sustained until 8 days after the denervation. This denervation-induced down-regulation of CREB phosphorylation might be one of the explanations for the transient up-regulation of PRiMA mRNA in denervated muscles. In addition, a higher level of active phosphorylated forms of CREB in the soleus could also explain why PRiMA is expressed at a lower level in slow twitch muscle.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The expression and distribution of AChE forms in mammalian muscles depend on the muscle fiber type, the motor nerve contact, and the contractile activity. Rat fast twitch muscles contain G1, G4, and A12 forms of AChE, in which A12 is largely predominant and exclusively localized at the nmjs. In slow twitch muscles, G1, A4, and A8 forms of AChE are relatively abundant and also found in extrasynaptic regions of the muscle fibers (24, 33), and the proportion of the G4 form is low. However, the roles of all of these AChE molecular forms and the processes by which they are produced in postsynaptic muscles remain a puzzle. Massoulié and co-workers (2, 33) proposed that the assembly of AChE oligomers could be a random process and would thus be determined by the relative abundance of AChET, ColQ, and PRiMA proteins in muscles, producing either A12, A8, A4, or G4 forms of AChE. In order to test this hypothesis, we provide a detailed analysis of PRiMA mRNA expression in cultured muscle and analyze the influence of different nerve-derived factors. As indicated by our current results in cultured C2C12 myotubes, there are two possible mechanistic pathways in regulating PRiMA mRNA. First, MRFs, including myogenin and MyoD, are able to suppress the expression of PRiMA and to cause G4 AChE down-regulation during myogenic differentiation. Second, nerve-derived factors, including muscular activity and CGRP, suppress the expression of PRiMA. This suppression is mediated by the phosphorylation of CREB. In addition, muscular activity-induced CREB phosphorylation is mediated by the activation of CaMKII. These results explain the normal physiological phenomenon that innervation reduces PRiMA and G4 AChE expression in muscle.


Figure 8
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  		FIGURE 8.
CREB is a crucial regulator in suppressing PRiMA expression. A, the cDNA encoding wild-type CREB was overexpressed in C2C12 myotubes. Different amounts of cDNA were used. Quantitative real time PCR was performed to reveal the mRNA expressions of AChET and PRiMA I (as in Fig. 3). Data are normalized and expressed as a percentage of the control (vector-transfected cultures). B, the cDNA encoding K-CREB was overexpressed in C2C12 myotubes before the application of various drugs: ACh (100 µM), KCl (20 mM), CGRP (1 µM), and Bt2-cAMP (3 mM). Two days later, total RNAs (5 µg) were extracted from the cultures, and quantitative real time PCR was performed to reveal the mRNA expressions of PRiMA I (top) and AChET (bottom). Data are normalized and expressed as a percentage of control (untreated culture). All values are mean ± S.E., n = 4, each with triplicate samples.

 
By using an antibody specific for PRiMA, we provide different lines of evidence to suggest that a significant portion of muscle G4 AChE is associated with PRiMA. The possibility of the existence of non-PRiMA-associated G4 (e.g. degraded products from the A12 enzyme) could not be eliminated in this study. In rodents, the level of G4 AChE is higher in fast twitch muscle than in slow twitch muscle, and this G4 AChE expression is regulated by the innervating nerve (16–18). These physiological phenomena could be explained partly by the regulation of PRiMA transcription in muscles. The high expression of PRiMA mRNA may explain the higher amount of G4 AChE in fast twitch muscles. Indeed, the tibialis muscle has an ~10-fold higher level of PRiMA mRNA than the soleus muscle has (Fig. 9A). In contrast, the low PRiMA expression in slow twitch muscles could result from the relatively high level of the active form of CREB (a suppressor of PRiMA expression) in muscles. The reason why phospho-CREB is higher in soleus is not known but could be related to the intrinsic muscular activity of slow twitch muscle fibers. CaMKII-mediated CREB phosphorylation does not totally account for this difference, because the total and phosphorylated forms of CaMKII seem to be relatively equal in both types of muscle fibers. Thus, other phosphorylators of high levels of CREB in slow twitch muscles should be considered.

In mature myotubes or innervated muscle fibers, ColQ-associated A forms of AChE are the predominant species of the enzyme, being localized at vertebrate nmjs to terminate cholinergic transmission (2, 34, 35). During myogenic differentiation of muscle, ColQ mRNA expression is increased (21, 24). A reciprocal regulation of PRiMA and ColQ gene transcriptions during myotube formation provides a regulatory mechanism to assemble the A form AChE preferentially rather than G4 AChE in muscle, in a precise and temporal manner. One of the underlying molecular mechanisms for these differential expressions of ColQ and PRiMA could be the control of transcription by MRFs during the onset of myogenesis (Fig. 5). Myogenin and MyoD repressed the transcription of PRiMA mRNA in cultured myotubes and reduced the proportion of G4 AChE, whereas the transcriptional activity of the COLQ gene could be activated by overexpression of myogenin and MyoD (21, 24). On the other hand, the up-regulation of AChET mRNA by myogenin or MyoD, as well as the promoter activity of the AChE gene (36), is essential to maintaining a sufficient amount of AChET for the production of the A form of AChE. E-box-responsive elements in the rat AChE promoter have been shown to be involved in myogenin-mediated activation (36). On the other hand, the upstream regulating elements of human PRiMA4 and COLQ (20) genes contain several putative E-box sites, and therefore MRFs might regulate the gene transcriptions in opposite manners.


Figure 9
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  		FIGURE 9.
Denervation-induced PRiMA expression is mediated by the phosphorylation of CaMKII and CREB in fast and slow muscles. A, total RNAs extracted from soleus and tibialis of rat were subjected to quantitative real time PCR to reveal the mRNA expressions of AChET and PRiMA I. Data are normalized and expressed as the ratio to basal reading (RNA in soleus is arbitrary set as 1). B, an ~3-mm portion of the sciatic nerve was cut from the upper thigh. Soleus and tibialis were collected on days 1, 2, 5, and 8 after denervation. Quantitative real time PCR was performed to reveal the mRNA expressions of PRiMA (left) and AChET (right) as in A. Data are normalized and expressed as a percentage of the control (day 0; no denervation). C, 50 µg of protein, from denervated muscles, was loaded per lane in the Western blot for probing total and phosphorylated forms of CaMKII and CREB. Muscles from sham-operated rats served as controls at different time points. D, the amount of phosphorylation from the blots in C was quantified by calibrated densitometry. Data are normalized and expressed as the percentage of the control (maximal values in each case). All values are mean ± S.E., n = 4, each with triplicate samples.

 
Our current results indicate a parallel regulation of PRiMA and G4 AChE in muscle, which strongly suggests a role of PRiMA in directing the formation of G4 AChE. The requirement of PRiMA in forming G4 AChE has been well characterized in transfected COS cells. By transfecting cDNAs encoding AChET and PRiMA in COS cells, Bon and co-workers (5, 37) have demonstrated that G4 AChE was the predominant species being formed in the transfected cells. Here, we extended her findings by showing that PRiMA and AChET can assemble autonomously in cultured myotubes. This G4 AChE assembly did not require the intracellular carboxyl terminus of PRiMA. A hypothesis has been proposed that the intracellular carboxyl terminus of PRiMA I, containing potential myristoylation and phosphorylation sites, is used to direct the localization of G4 AChE at the plasma membranes of neurons and muscles (6). In addition to helping in the G4 assembly, the role of PRiMA in directing the localization of G4 AChE in the synapses should also be considered. This notion creates a reason to find the synaptic and extrasynaptic localization of G4 AChE at the nmjs of either fast or slow twitch muscles.

CGRP knock-out mutant mice showed structurally and ultrastructurally identical nmjs, as compared with wild-type animals; however, other signals might compensate for the absence of CGRP in directing the formation or maintenance of the nmjs (38). In contrast with the results of the knock-out study, emerging lines of evidence suggest that CGRP is still an excellent candidate to be a neuron-derived factor in directing the formation and/or the maintenance of postsynaptic specializations, in particular the regulation of AChE in muscle. Several studies have suggested an AChE-regulating role for CGRP in muscle, including the regulation of AChET in rodents (14) and chickens (13), the induction of ColQ mRNA (23), the modification of AChE molecular forms (32), and the suppression of PRiMA (Fig. 6) and G4 AChE (31). On the other hand, functional receptor(s) for CGRP have been identified in the postsynaptic muscle fibers in chickens (39), quail (32), and rats,5 in which the CGRP receptors trigger downstream cAMP-dependent signals upon activation. These various lines of evidence converge to establish CGRP as a key regulator of AChE at the nmjs.


   FOOTNOTES
 
* This work was supported by Research Grants Council of Hong Kong Grants HKUST 6283/03M, 6237/04M, 6404/05M, 6419/06M, and 3/03C (to K. W. K. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a Croucher Foundation Scholarship. Back

2 Recipient of a visiting professorship at Ecole Normale Supérieure in 2004 and 2006. To whom correspondence should be addressed. Tel.: 852-2358-7332; Fax: 852-2358-1559; E-mail: BOTSIM{at}UST.HK.

3 The abbreviations used are: nmj, neuromuscular junction; AChE, acetylcholinesterase; PRiMA, proline-rich membrane anchor; CGRP, calcitonin generelated peptide; MRF, myogenic regulatory factor; CREB, cAMP-responsive element-binding protein; HEK, human embryonic kidney; CaMKII, calcium/calmodulin-dependent protein kinase II; ACH, acetylcholine chloride; Bt2-cAMP, N6, O2'-dibutyryl-cAMP. Back

4 H. Q. Xie, R. C. Y. Choi, K. Wing Leung, N. L. Siow, L. W. Kong, F. T. C. Lau, H. Benjamin Peng, and K. W. K. Tsim, unpublished data. Back

5 A. K. L. Ting and R. C. Y. Choi, unpublished result. Back



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