Differential regulation of PRiMA expression in assembling G4-AChE in muscle

28 Pages·2007·0.5 MB·English

by  Roy & Fion

JBC Papers in Press. Published on February 26, 2007 as Manuscript M608265200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M608265200

REGULATION OF A TRANSCRIPT-ENCODING THE PROLINE-RICH
MEMBRANE ANCHOR (PRIMA) OF GLOBULAR MUSCLE
ACETYLCHOLINESTERASE: THE SUPPRESSIVE ROLES OF MYOGENESIS AND
INNERVATING NERVES*

Heidi Q. Xie, Roy C. Y. Choi, K. Wing Leung, Nina L. Siow, Ling W. Kong,
Faye T. C. Lau, H. Benjamin Peng and Karl W. K. Tsim
The Department of Biology and the Molecular Neuroscience Center,
The Hong Kong University of Science and Technology, Clear Water Bay Road,
Hong Kong, China
Running Title: Regulation of PRiMA in muscle
Address correspondence to: Prof. Karl W.K. Tsim, Department of Biology, The Hong Kong University
of Science and Technology, Clear Water Bay Road, Kowloon, Hong Kong SAR, China,
Tel. [phone]; Fax. [phone]; E-mail: [email protected] D
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The transcriptional regulation of regulatory factors, myogenin and myoD, the ed
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proline-rich membrane anchor (PRiMA), expressions of PRiMA and G AChE in m
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an anchoring protein of tetrameric globular cultured myotubes were markedly reduced. ttp://w
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form acetylcholinesterase (G4 AChE), was In addition, calcitonin gene-related peptide w
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revealed in muscle during myogenic (CGRP), a known motor neuron-derived c.o
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differentiation under the influence of factor, and muscular activity were able to b/
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innervation. During myotube formation of suppress PRiMA expression in muscle; the e
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C2C12 cells, the expression of AChET suppression was mediated by the n Ja
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protein and the enzymatic activity were phosphorylation of a cAMP-responsive a
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dramatically increased, but the level of G4 element-binding protein (CREB). In 3, 2
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AChE was relatively decreased. This G accordance with the in vitro results, sciatic 9
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AChE in C2C12 cells was specifically nerve denervation transiently increased the
recognized by anti-PRiMA antibody, expression of PRiMA mRNA and decreased
suggesting the association of this enzyme the phosphorylation of CREB, as well as its
with PRiMA. RT-PCR analysis revealed activator calcium/calmodulin-dependent
that the level of PRiMA mRNA was reduced protein kinase II (CaMK II), in muscles.
during the myogenic differentiation of Our results suggest that the expression of
C2C12 cells. Over expression of PRiMA in PRiMA, as well as PRiMA-associated G
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C2C12 myotubes significantly increased the AChE, in muscle is suppressed by muscle
production of G AChE. The regulatory factors, muscular activity and
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oligomerization of G AChE, however, did nerve-derived trophic factor(s).
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not require the intracellular cytoplasmic tail
of PRiMA. After over expressing the muscle During cholinergic transmission at
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Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
neuron-to-neuron synapses in the central and may contribute to its development (8).
nervous system or neuromuscular junctions Thus, G AChE may have distinct functions in
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(nmjs) in the peripheral nervous system, different tissues.
acetylcholinesterase (AChE; EC 3.1.1.7) plays Although G is not the major form of
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a crucial role in terminating the synaptic AChE in muscle, its existence is tightly
transmission by hydrolyzing the controlled. Several studies have revealed that
neurotransmitter acetylcholine. Depending on the level of G AChE is controlled by the
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alternative splicing in the 3’ region of the dynamic activity of skeletal muscles. In
primary transcript, AChE exists in different mammals, fast-twitch muscles contain a high
molecular forms (1). This process generates amount of G , whereas slow-twitch muscles
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different subunits that contain the same contain a much smaller amount (9). Alteration
catalytic domain but with distinct carboxyl of the G AChE level after muscle denervation
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termini (1, 2). In mammals, the AChE variant strongly suggests a critical role of motor
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produces a soluble monomer that is up nerves in G AChE regulation (2, 10). The D
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regulated in the brain during stress (3); the motor nerves may achieve this regulation by nlo
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AChEH variant produces a GPI-anchored two distinct mechanisms: release of trophic ed fro
dimer that is mainly expressed in blood cells; factor and nerve-evoked electrical activity. m
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the AChET variant is the only subunit Among the known nerve-derived trophic ttp://w
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expressed in the brain and muscle. AChET factors, calcitonin gene-related peptide w
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subunits form non-amphiphilic tetramers with (CGRP), a neuropeptide with 37 amino acids, c.o
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a collagen tail (ColQ) as asymmetric AChE which has been identified in spinal cord motor b/
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(A AChE) in muscle, and also form neurons (11), exerts an innervation-like effect e
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amphiphilic tetramers associated with a to suppress G4 AChE when applied in muscles n Ja
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proline-rich membrane anchor (PRiMA) as (12). In addition, CGRP regulates the synthesis a
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globular form AChE (G4 AChE) in brain and of the AChET subunit (13-15) and of 3, 2
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muscle (4, 5). acetylcholine receptors (AChR) (11). On the 9
G AChE has been found in other hand, exercise induces a marked change
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mammalian tissues, including brain, muscle in the level of this enzyme form, without
and heart (2); its expression pattern exhibits a modification of other molecular species
close resemblance to PRiMA RNA expression (16-18). Unfortunately, G AChE was
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(6). In addition to the key role of AChE in analyzed only by sedimentation, and the
cholinergic function, the correct orientation of expression of PRiMA, the only G -specific
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AChE catalytic subunits at the cell surfaces of component, has not been studied under
certain neurons, targeted by PRiMA, is physiological conditions.
proposed to be required for neurite outgrowth In this study, we sought to identify
(7). Additionally, G AChE in the brain is PRiMA-associated G AChE in cultured
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related with amyloid plaques and C2C12 muscle cells and to analyze the
neurofibrillary tangles in Alzheimer's disease expression of mRNAs encoding PRiMA, as
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well as AChE , in cultured C2C12 cells during fibroblast cell line was obtained from the
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myogenic differentiation, the influence of ATCC and cultured in DMEM supplemented
nerve-derived factors and muscular activity, with 10% FBS at 37 oC in a water-saturated
and the effect of denervation in fast-twitch and 5% CO incubator.
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slow-twitch muscles. Our results indicate that DNA construction and transfection - cDNAs
myogenic regulatory factors (MRFs), muscular encoding full-length mouse PRiMA (PRiMA I),
activity and CGRP suppress the expression of and a C-terminal truncated mutant (PRiMA
PRiMA, probably mainly by activating the I ; obtained by deleting the C-terminal
ΔC-term
CREB (cAMP-responsive element binding region, 122-153) were tagged by FLAG
protein) transcription factor. In addition, the epitope (obtained by inserting a FLAG epitope
production of G AChE in muscle is shown to of DYKDE at position 36 between the putative
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be controlled by the level of PRiMA signal sequence and the N-terminus) in
expression. pER-BOS mammalian expression vector (6).
The mouse myogenin and myoD cDNAs were D
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Experimental Procedures described in Lee et al. (20). Vectors expressing nlo
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the CREB (wild-type) and K-CREB (inactive ed
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Cell cultures - The mouse C2C12 muscle cell mutant) cDNAs were purchased from m
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line was obtained from the American Type Clontech Laboratories (Mountain View, CA). ttp://w
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Culture Collection (ATCC, Manassas, VA). The cDNA encoding the constitutively active w
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Undifferentiated C2C12 myoblasts were form of rat calcium/calmodulin-dependent c.o
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maintained in Dulbecco’s Modified Eagle’s protein kinase II-γ (CaMKII-γ) was subcloned b/
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Medium (DMEM) supplemented with 20% into pCS2MT vector (21). Transient e
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fetal bovine serum (FBS) and incubated at 37 transfection of myoblasts with the cDNA n J
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oC in a water-saturated 5% CO incubator. All construct was performed with a ua
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reagents for cell cultures were from Invitrogen Lipofectamin-plus reagent (Invitrogen), 3, 2
0
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(Carlsbad, CA). Myogenic differentiation was according to the manufacturer’s instructions. 9
induced as previously described (19). In brief, The transfection efficiency was consistently
the cultured myoblasts were allowed to grow 30-40% in the C2C12 myoblasts. The
in DMEM with 10% FBS until they were transfection in the cultured HEK293T
confluent, and then they were changed to fibroblasts was done by calcium phosphate
DMEM with 2% heat-inactivated horse serum precipitation as described previously (15).
to induce differentiation. In the myogenesis Production and purification of anti-PRiMA
studies, cell lysates were collected on each day polyclonal antibody – The mouse PRiMA
starting from the first day of induction (day 0) (from amino acids 114-153)-GST fusion
to the eighth day (day 7), and the extracts were protein was expressed in BL21 (DE3) pLysE E.
stored at -80 oC. The drug treatments were coli (Invitrogen) and purified by glutathione
carried out on four-day-old myotube cultures. bead chromatography (Amersham Biosciences,
The human embryonic kidney (HEK) 293T Piscataway, NJ) according to the
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manufacturer’s instructions. After digestion by instructions of the Animal Care Facility at
thrombin (Sigma, St. Louis, MO), the PRiMA Hong Kong University of Science and
(114-153) antigen was purified by Superdex Technology. Soleus and tibialis muscles were
75 10/300 gel filtration chromatography collected on days 1, 2, 5 and 8 after
(Amersham Biosciences). Polyclonal denervation. Muscle samples were frozen in
antibodies were raised in a 2 kg male New liquid nitrogen immediately after dissection
Zealand white rabbit by immunization with and stored at -80 oC before the RNA or protein
750 μg antigen, mixed with an equal volume extraction. Control experiments were
of complete Freund’s adjuvant (Sigma). The performed by sham operations on the same
immunization was carried out with the same muscles of different rats.
amount of antigen three times within 1 month. Real-time quantitative PCR - Total RNA from
The anti-PRiMA serum was collected and either C2C12 cultures or rat tissues was
purified by protein G Sepharose (Amersham isolated by TRIzol reagent (Invitrogen), and 5
Biosciences) according to the manufacturer’s μg of RNA was reverse-transcribed by D
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instructions. The amount of purified antibody Moloney Murine Leukemia Virus Reverse nlo
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was determined spectrophotometrically. Transcriptase (Invitrogen), according to the ed
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Drug treatments – Four-day-old cultured manufacturer’s instructions. Real-time PCR of m
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myotubes were treated with either PRiMA, AChET and GAPDH transcripts was ttp://w
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acetylcholine chloride (ACh; 10 and 100 μM), performed on equal amounts of w
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depolarizing agent potassium chloride (KCl; reverse-transcribed products, using SYBR c.o
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10 or 20 mM), Ca2+ ionophore A23187 (0.2 or Green Master mix and Rox reference dye, b/
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0.5 μM), CGRP (1 μM) or N6, according to the manufacturer’s instructions ue
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O2’-dibutyryl-adenosine 3’: 5’-cyclic (Applied Bioscience, Foster City, CA). The n J
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monophosphate (Bt -cAMP; 0.3 and 1 mM) primers were: 5’-TCT GAC TGT CCT GGT a
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for 2 days. Pre-treatment with KN62 (20 μM; CAT CAT TTG CTA C-3’ and 5’-TCA CAC 3, 2
0
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an inhibitor of CaMKII) was done for 3 hours CAC CGC AGC GTT CAC-3’ for mouse 9
before the drug application. In PRiMA I and II (Genbank number NM 133364
phosphorylation analyses, myotube cultures and NM 178023); 5’-CTG GGG TGC GGA
were serum-starved for 3 hours before the drug TCG GTG TAC CCC-3’ and 5’-TCA CAG
application. All of the drugs were purchased GTC TGA GCA GCG TTC CTG-3’ for mouse
from Sigma. AChE (23), 5’-AAC GGA TTT GGC CGT
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Sciatic nerve denervation - Two-month-old ATT GG-3’ and 5’-CTT CCC GTT CAG CTC
Sprague-Dawley rats weighing ~250 g were TGG G-3’ for mouse and rat
anaesthetized by isoflurane. Denervation was glyceraldehyde-3-phosphate dehydrogenase
performed by removing a ~3 mm portion of (GAPDH; 21). The SYBR green signal was
the sciatic nerve located around the upper detected by a Mx3000ptm multiplex
thigh, by an aseptic surgical technique (22). quantitative PCR machine (Stratagene, La
Rats were sacrificed according to the Jolla, CA). The transcript expression levels
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were quantified by using the Ct value method calibration plot constructed from a parallel gel
(24), where values were normalized to with serial dilutions of one of the samples. In
GADPH as an internal control in the same the immunofluorescent analysis, the cDNA
sample. The PCR products were analyzed by transfected HEK293T cells, after 2 days of
gel electrophoresis, and the specificity of transfection, were fixed by 4%
amplification was confirmed by the melting paraformaldehyde (PFA) and 4% sucrose in
curves. PBS for 5 min, followed by 50 mM
Immunochemical analysis - C2C12 cultures, ammonium chloride (NH Cl) treatment for 25
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cDNA-transfected HEK 293T cultures, muscle min. Cultures were permeabilized and blocked
and brain tissues were homogenized in a lysis by 5% FBS, 0.1% Triton X-100 in PBS for for
buffer (10 mM HEPES, pH 7.5, 1 M NaCl, 1 1 hour at room temperature. Anti-PRiMA
mM EDTA, 1 mM EGTA, 0.5% Triton X-100 antibody (2 μg/ml) and anti-FLAG antibody
and 1 mg/ml bacitracin) followed by (dilution 1:500) were applied to the cells for
centrifugation at 12,000 g for 20 min at 4 oC. 16 hours at 4 °C followed by the D
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Protein samples were denatured at 100oC for 5 corresponding Alexa 488-conjugated nlo
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min in a buffer containing 1% SDS and 1% anti-rabbit secondary antibody for 2 hours at ed
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dithiothreitol and separated by 8% or 12% room temperature. The cells were dehydrated m
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SDS-polyacrylamide gel electrophoresis. In serially with 50%, 75%, 95% and 100% ttp://w
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the western blot analysis, we used anti-PRiMA ethanol and mounted with a fluorescence w
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polyclonal antibody (purified at 0.5 µg/ml), mounting medium (DAKO, Carpinteria, CA). c.o
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anti-AChE antibody (1:5,000; BD The samples were then examined by a Leica b/
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Biosciences, San Jose, CA), anti-FLAG confocal microscope with Ex 488/Em 505-550 e
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antibody (1:1,000; Sigma), anti-myogenin and nm for green color. n J
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anti-myoD antibodies (1:1,000; Santa Cruz Sucrose density gradients - Separation of the a
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Biotechnology, Inc., Santa Cruz, CA), various molecular forms of AChE was 3, 2
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anti-α-tubulin antibody (1:5,000; Sigma), performed by sucrose density gradient analysis, 9
anti-phospho-CaMKII and anti-total CaMKII as described previously (15). In brief, sucrose
antibodies (1:1,000; Upstate, Billerica, MA), gradients (5 and 20%) in a lysis buffer (10 mM
anti-phospho-CREB and anti-total CREB HEPES, pH 7.5, 1 M NaCl, 1 mM EDTA, 1
antibodies (1:1,000; Cell Signaling Technology, mM EGTA, and 0.5% Triton X-100) were
Danvers, MA). The immune complexes were prepared in 12 ml polyallomer
visualized using the enhanced ultra-centrifugation tubes with a 0.4 ml
chemiluminescence (ECL) method cushion of 60% sucrose on the bottom. Cell
(Amersham Biosciences). The intensities of extracts (0.2 ml) mixed with sedimentation
the bands in the control and stimulated markers (alkaline phosphatase, 6.1S;
samples, run on the same gel and under strictly β-galactosidase, 16S) were loaded onto the
standardized ECL conditions, were compared gradients and centrifuged at 38,000 rpm in a
on an image analyzer, using, in each case, a Sorvall TH 641 rotor at 4 oC for 16 hours.
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Approximately 45 fractions were collected and RESULTS
the AChE enzymatic activity was determined
according to the method described by Ellman Regulation of G AChE and PRiMA during
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(25) with the modification of adding 0.1 mM myogenic differentiation
tetra-isopropylpyrophosphoramide Cultured mouse C2C12 cells were
(iso-OMPA), an inhibitor of used as a model system for determining the
butyrylcholinesterase, to each fraction. The expression profile of AChE during myogenic
absorbance at 410 nm was recorded as a differentiation. In the absence of serum,
function of the reaction time. The amount of C2C12 myoblasts were allowed to undergo
the various AChE forms was determined by fusion, forming multi-nucleated myotubes.
summation of the enzymatic activities The western blots showed that AChE protein
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corresponding to the peaks of the (~68 kDa) increased by ~8.5-fold in C2C12
sedimentation profile. In the cells during myogenesis, while the loading
immunoprecipitation of G AChE by control, α-tubulin (~55 kDa), remained D
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anti-PRiMA antibody, brain, muscle and unchanged (Fig. 1A). In line with the protein nlo
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C2C12 cell extracts (1 ml in 10 mM HEPES, profile, the enzymatic activity of AChE ed
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pH 7.5, 1 M NaCl, 1 mM EDTA, 1 mM EGTA, dramatically increased (~12-fold) from the m
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0.1% Triton X-100 and 1 mg/ml bacitracin) myoblast to the myotube stage (Fig. 1A), in ttp://w
were incubated for 4 hours at 4 oC with agreement with previous results (19). ww
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purified anti-PRiMA antibody (10 μg/ml). Sucrose density gradient analysis was c.o
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Then, 50 μl of washed protein-G agarose gel used to investigate the AChE molecular forms b/
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(Santa Cruz Biotechnology) was added and found during the process of muscle e
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incubated for 1 hour at 4 oC. After differentiation. At the myoblast stage (day 0), n J
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centrifugation, the supernatants were loaded AChE existed predominantly in the G form, a
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on sucrose gradients for sedimentation together with trace amounts of the G1 form 3, 2
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analysis. (Fig. 1B). When the myoblasts fused to form 9
Other assays - Protein concentrations were myotubes on day 4, the relative amount of G
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measured routinely using Bradford's method AChE was reduced and G AChE became the
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(26) with a kit from Bio-Rad Laboratories predominant form. A small amount of A
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(Hercules, CA). Statistical tests were run on AChE (ColQ-associated) appeared in mature
the PRIMER program, version 1 (Primer of myotubes on day 7 (Fig. 1B). By quantifying
Biostatistics, S. A. Glantz, McGraw-Hill, Inc. the absolute amount of G /G AChE in muscle
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1988): differences from basal or control values during differentiation, we found that the
(as shown in the plots) were classified as amount of G increased by ~4-fold on day 4
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significant [*] for p<0.05, [**] and p<0.01 and (as well as on day 7) of myotube formation;
highly significant [***] for p<0.001. however, the increase in G was more robust
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(over 100-fold) (Fig. 1C). These results reveal
that while AChE protein and enzymatic
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activity are up-regulated during the myogenic extracts was depleted by the antibody
differentiation process, the relative proportion treatment with ~70% depletion in brain, ~40%
of G AChE decreases. depletion in muscle and ~40% depletion in
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Besides in brain enzymes, the cultured C2C12 cells. Clearly, the depletion
association of PRiMA with G AChE has not was more robust in the brain extracts. The G
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been identified biochemically in other tissues. enzyme in both cases was not affected by this
We generated anti-PRiMA polyclonal antibody antibody. These results suggest that a major
to address this gap. In FLAG-tagged PRiMA part of G AChE in muscle is associated with
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cDNA transfected HEK293T cells, both PRiMA. The identity of the rest of G AChE
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anti-PRiMA and anti-FLAG antibodies was not further analyzed.
recognized a protein band of size ~20 kDa, A reduction in the expression of
corresponding to the predicted size of PRiMA, PRiMA may explain the reduction in G AChE
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in our western blots (Fig. 2A left panel). The during the myogenic differentiation process.
recognition could be blocked by the According to Perrier et al. (27), two splicing D
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pre-incubation of anti-PRiMA antibody with variants of PRiMA mRNAs are generated nlo
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excess amounts of PRiMA peptides. This did from the PRiMA gene to produce different ed
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not occur with anti-FLAG antibody (Fig. 2A proteins (PRiMA I and PRiMA II; Fig. 3A). m
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right panel). In parallel, the antibody PRiMA I mRNA possesses exons 4 and 5 and ttp://w
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recognition was further confirmed in the produces a 40-residue-long intracellular w
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immunofluorescent staining of FLAG-tagged cytoplasmic tail, while PRiMA II mRNA c.o
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PRiMA cDNA transfected cells (Fig. 2B). possesses exons 4, 4b and 5, resulting in a b/
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These results indicated the specificity of our short intracellular motif (Fig. 3A). To e
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anti-PRiMA antibody. differentiate these two PRiMA isoforms, n J
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By using the anti-PRiMA antibody in RT-PCR was performed by specific primers a
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the western blots, a band of ~20 kDa was located in exon 4 and exon 5. In C2C12 3, 2
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recognized in the extracts derived from C2C12 cultures, a large amount of PRiMA I was 9
myoblasts and myotubes: the expression level found, while PRiMA II was barely detectable
was relatively higher in myoblasts (Fig. 2C (Fig. 3B). Similarly, both the tibialis (fast) and
left panel). In addition, PRiMA was also soleus (slow) muscles predominantly
detected in muscle (tibialis). Serving as a expressed PRiMA I (Fig. 3B). In adult rat
control, the brain extract showed similar band brain, both isoforms of PRiMA exist, which
recognition. Again, the antibody recognition means that rat brain can serve as a positive
was blocked by PRiMA peptides in all cases control, as reported previously (27).
(Fig. 2C right panel). To understand the Regulations in the level of PRiMA
association of PRiMA with G AChE, the brain, mRNA were determined by quantitative
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muscle and C2C12 cell extracts were real-time PCR analysis using the same sets of
immunoprecipitated by anti-PRiMA antibody. primers as in Fig. 3B. This showed that
As shown in Fig. 2D, the G AChE in the PRiMA I was expressed at high levels in the
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myoblast stage; the expression declined after of the cytoplasmic tail of PRiMA I in G
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the onset of differentiation; and finally it AChE oligomerization, this tail region was
reached a low level in the myotube stage (Fig. deleted to form PRiMA I (Fig. 4C). This
ΔC-term
3C). The level of PRiMA mRNA was reduced truncated cDNA construct, resembling PRiMA
by at least 50% during myogenesis. In contrast, II, was co-expressed with AChE cDNA in
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the level of AChE mRNA increased C2C12 cultures. The truncated PRiMA I
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dramatically (~120 fold) from the myoblast markedly increased the production of G
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stage to the myotube stage, in agreement with AChE (Fig. 4C) in the same manner as the full
the up regulation of AChE protein and the length PRiMA I. These results clearly indicate
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enzymatic activity. On the other hand, PRiMA that PRiMA I and PRiMA II are able to direct
II mRNA was maintained at a low level the assembly of AChE into G AChE in
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throughout the entire differentiation process; muscle, and the intracellular cytoplasmic tail
its expression was not further investigated. of PRiMA I does not required in this
Therefore, we refer to PRiMA I as PRiMA oligomerization process. Thus, the decrease of D
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hereafter, unless otherwise specified. G4 AChE during myogenic differentiation nlo
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The down regulation of PRiMA could be mainly attributed to the down ed
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mRNA in C2C12 cultures during myogenesis regulation of PRiMA. m
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is consistent with the observed reduction of G4 ttp://w
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AChE. In order to determine the possible role Myogenic regulatory factors regulate w
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of PRiMA in directing the formation of G4 PRiMA expression c.o
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AChE in muscle, C2C12 myoblasts were In order to elucidate the mechanism b/
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co-expressed with cDNAs encoding AChE that suppresses the transcription of the PRiMA e
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and PRiMA I; the molecular forms of AChE gene during muscle differentiation, we n J
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were analyzed subsequently at the myotube investigated the possible role of MRFs. a
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stage. Over expression of AChET in the Among different muscle-specific transcription 3, 2
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cultures produced mostly G AChE, with factors, myogenin (28) and myoD (29) have 9
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minor amounts of G and A AChE (Fig. 4A), been shown to play roles in the early phase of
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in agreement with the endogenous expression myotube formation. During C2C12
of AChE in the myotubes. Over expression of differentiation, myogenin (~36 kDa) and
PRiMA together with AChE markedly myoD (~38 kDa) were found to be induced,
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increased G AChE in the myotubes (Fig. 4B), reaching a maximal expression at the onset of
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indicating that the oligomerization of G AChE the differentiation process, then declining after
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was directed by PRiMA. Over expression of 4 days of fusion, and subsequently remaining a
PRiMA alone did not significantly increase the low level in mature myotubes (Fig. 5A).
amount of G AChE in the cultures; this might To determine the regulatory role of
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be due to the limited supply of AChE for G MRFs on PRiMA mRNA expression, C2C12
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AChE assembly. myoblasts were transfected with cDNAs
To determine the possible requirement encoding myogenin and myoD and allowed to
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form myotubes. Over expression of myogenin myotube cultures, ACh (10 and 100 μM),
and myoD decreased the expression of PRiMA depolarizing agent KCl (10 and 20 mM), and
mRNA to ~50%, as compared with the mock Ca2+ ionophore A23187 (0.2 and 0.5 μM) were
control pcDNA3 (Fig. 5B). In contrast, the applied to the myotube cultures for 2 days and
level of AChE mRNA increased by over then the expressions of PRiMA and AChE
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expressing myogenin and myoD in muscle mRNAs were determined. Compared with the
cultures: the increase of AChE protein was controls, all the drug treatments reduced the
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correlated with an increase of enzymatic expression of PRiMA mRNA (Fig. 6A). This
activity by ~60%. These observations are in down regulation effect, induced by muscular
line with the notion that MRFs suppress activity, was also observed in the gene
PRiMA expression during the early stages of transcription of AChE . These results suggest
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myogenesis. that nerve-evoked muscular activity provides a
We also analyzed the molecular forms uniform signal across the entire muscle fiber to
of AChE in myogenin or myoD reduce the expressions of PRiMA and AChE . D
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cDNA-transfected myotubes. The CGRP, a nerve-derived trophic factor, nlo
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pcDNA3-transfected myotubes expressed is able to trigger an accumulation of ed
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mostly G and G AChE with a minor portion intracellular cAMP, which has been m
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of A12 AChE (Fig. 5C). When myogenin or demonstrated to regulate the synthesis and ttp://w
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myoD was over expressed in the myotubes, formation of G4 AChE in muscles (13-14, 31). w
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the total amount of G4 AChE within the Total RNAs were extracted from CGRP- (1 c.o
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transfected myotubes was reduced by over μM) or Bt -cAMP- (1 mM and 3 mM) treated b/
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50%. In contrast, the amount of A AChE was cultures for quantitative real-time PCR e
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increased by ~50% under the effects of the analysis. The amount of PRiMA mRNA was n J
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MRFs over expression (Fig. 5C and D). The reduced to ~30% by CGRP or Bt -cAMP (Fig. a
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reduction of G4 AChE was probably due to the 6A). Expression of AChET mRNA was also 3, 2
0
1
decrease of PRiMA expression under the reduced to ~50% in the drug-treated samples, 9
control of the over-expressed MRFs. as reported previously (19). In parallel with
the muscular activity- and CGRP-induced
Muscular activity and CGRP suppress PRiMA down regulation, the amount of G
4
PRiMA expression AChE in cultured myotubes was selectively
In vertebrate nmjs, the innervated decreased by these treatments: the reduction of
motor axon provides two types of anterograde G AChE was over 40% (Fig. 6B and C). In
4
signals, ACh-induced muscular activity and contrast, the expression of the total A AChE
12
nerve-derived factors, to control the remained almost unchanged.
expression of post-synaptic genes in muscle.
Muscular activity is known to suppress AChR CREB phosphorylation mediates PRiMA
expression via intracellular Ca2+ and CaMKII suppression
(22, 30). To mimic this muscular activity in The phosphorylation of CREB is one
9
of the downstream signals in CREB phosphorylation. In cultured myotubes,
CGRP/cAMP-induced signaling cascades and application of activity-inducing agents (ACh
has been demonstrated to be a key regulator in and A23187) induced a sustained
suppressing the expression of AChE in phosphorylation of CaMKII (~50 kDa) that
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muscle (15). Here, we determined its possible was at least 5-fold stronger (Fig. 7A and B).
role in PRiMA suppression. In serum-starved The total amount of CaMKII remained
myotubes, application of CGRP at 1 μM unchanged at different time intervals.
induced the phosphorylation of CREB (~43 Additionally, the activity-induced CREB
kDa), which was recognized by an phosphorylation could be fully blocked by the
anti-phospho-CREB antibody (Fig. 7A). We pre-treatment with KN62, a CaMKII inhibitor,
observed a transient CGRP-induced CREB in cultured myotubes (Fig. 7C). To confirm the
phosphorylation, with a peak of activation role of CaMKII in phosphorylating CREB, we
(~6-fold) 10 min after CGRP challenge. The over expressed an active form of CaMKII in
total amount of CREB at ~43 kDa remained cultured myotubes; this over expression D
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unchanged at different time intervals. Similarly, induced an activation of CREB (Fig. 7D). nlo
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application of muscular activity-inducing These results, therefore, suggest that CREB ed
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agents (ACh and A23187) also induced CREB may act as one of the downstream effectors for m
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phosphorylation: the phosphorylation was CaMKII activation in muscle. ttp://w
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sustained for a longer time with these agents In cultured myotubes, over expression w
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and the peak occurred 10-15 min after the drug of CREB decreased AChET and PRiMA c.o
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challenge (Fig. 7 A and B). Although ACh mRNAs in a dose-dependent manner; the b/
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should be rapidly hydrolyzed, its effect on reduction was more significant for PRiMA e
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CREB phosphorylation peaked at 10 min after mRNA (Fig. 8A) than for AChET. The role of n Ja
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the treatment. This result agrees with the role CREB in directing the suppression of PRiMA a
ry
1
that ACh plays in affecting the formation of expression by CGRP and muscular 3, 2
0
1
AChE forms. activity-inducing agents was further 9
These results are consistent with the demonstrated by using a dominant negative
fact that CGRP induced CREB mutant of CREB (K-CREB). Over expression
phosphorylation. The CGRP-induced CREB of K-CREB in cultured C2C12 myotubes
phosphorylation is mediated by a markedly reduced the decrease of PRiMA
cAMP-dependent signaling pathway, via a induced by ACh, KCl, CGRP, and Bt -cAMP
2
CGRP receptor complex on the muscle surface (Fig. 8B). These results support the argument
(15, 32). In contrast, the mechanism of that CREB plays a key role in the regulation of
muscular activity-induced CREB PRiMA expression in muscle.
phosphorylation has not yet been determined. The suppression of PRiMA expression
CaMKII is one of the downstream effectors of by CREB could be further confirmed by
the muscular activity-inducing agents (22, 30) comparing the expression of PRiMA mRNA in
and therefore could be responsible for the fast-twitch (tibialis) and slow-twitch (soleus)
10