AN ABSTRACT OF THE THESIS OF
Mei-Chuan Wang for the degree of Master of Science in Animal Science presented on
December 2, 1997.
Title: Effects of Ciliary Neurotrophic Factor (CNTF) on Protein
Turnover in Cultured L8 Rat Muscle Cells.
Abstract approved:
Redacted for Privacy
Neil E. Forsberg
Skeletal muscle proteins are the largest amino acid pool in animal body and are
continuously degraded and resynthesized. Dozens of factors have been shown to influence
the balance between synthesis and degradation and thereby regulate muscle growth and
function. It is now know that one of the major regulatory bases of muscle metabolism is
neuron-muscle interaction. A neurogenic factor, ciliary neurotrophic factor (CNTF), is
proposed to exert myotrophic actions and could possible be a mediator of neuron-muscle
interactions.
The goal of this study was to investigate the effects of CNTF on muscle protein
turnover in an in vitro system. CNTF was applied to differentiated cultured muscle cells (L8
cell line). Radiochemical labeled amino acids were added to the culture medium to determine
the rate of incorporation or release by the cells as indexes of protein synthesis and protein
degradation, respectively. Total protein was measured as an index of change in total protein
accretion.
Twelve hours of CNTF treatment increased myofibrillar protein synthesis by 10%.
However, longer CNTF treatment (24 hours) reduced non-myofibrillar protein synthesis.
CNTF (1 ng/ml) decreased protein degradation but higher doses (20 ng/ml) accelerated
protein degradation. These changes in protein turnover resulted from changes in the
myofibrillar protein fraction rather than from changes in turnover of the non-myofibrillar
fraction. The change in protein synthesis and protein degradation resulted in an increase in
total protein accretion of about 6%. Compared with the myotrophic studies on the effects
of CNTF in vivo, the action of CNTF were relatively small. Reverse transcription polymerase
chain reaction (RT-PCR) analysis showed that CNTF receptor alpha subunit (CN1kRa)
mRNA expression is lower than which is expressed in muscle tissue. This could explain the
reason why CNTF exerted smaller effects in vitro compared to those reported in vivo.
Overall, CNTF exerts a small but statistically significant anabolic actions in cultured
skeletal muscle and the actions were highly dose-dependent. The limited action of CNTF in
vitro may be related to its low receptor density in the L8 cell (compared to in vivo). Because
actions may be highly dose-dependent, a challenging series of studies are ahead for anyone
wishing to identify the signal transduction mechanisms which account for CNTF's actions.
Effects of Ciliary Neurotrophic Factor (CNTF) on Protein Turnover in
Cultured L8 Rat Muscle Cells
by
Mei-Chuan Wang
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented December 2, 1997
Commencement June 1998
Master of Science thesis of Mei-Chuan Wang presented on December 2, 1997
APPROVED:
Redacted for Privacy
Major Professor, representing Animal Sciences
Redacted for Privacy
Head or Chair of Department of Animal Science
Redacted for Privacy
Dean of Graduffte School
I understand that my thesis will become part of the permanent collection of Oregon State
University libraries. My signature below authorizes release of my thesis to any reader upon
request.
Redacted for Privacy
Mei-Chuan Wang, Author
ACKNOWLEDGMENT
I
sincerely appreciate my major professor, Dr. Forsberg, for his guidance,
encouragement and financial support throughout my program. He provided me a great
opportunity to expand my knowledge and he inspired my enthusiasm for research. I also
appreciate Dr. Philip Whanger for his advice and for letting me use his laboratory equipment.
Thanks are extended to my other committee members, Dr. Steve L. Davis and Dr. Masakazu
Matsumoto, for their precious suggestions in completing my thesis.
I greatly appreciate the many others who contributed to this project, Mr. Mike
Beilstein for technical discussion and editing my thesis; Dr. Qiuping Gu for assistance in
molecular biology techniques; Dr. Bor-rung Ou for valuable suggestion and patient teaching.
I am grateful for my colleagues, Jing Huang, Yung-Hae Kim, Roustem Nabioulem, Juntipa
Purintrapiban Yoji Ueda Pai-yen Wu and Ying-Yi Xiao, for the friendship, encouragement,
and scientific discussions.
Finally, I give special thanks to all the staff in the Department of Animal Sciences and
Oregon State University, for providing me an excellent learning and working environment
during my studies.
TABLE OF CONTENTS
Page
INTRODUCTION
1
LITERATURE REVIEW
6
MATERIALS AND METHODS
29
RESULTS
39
DISCUSSION
53
CONCLUSION
60
BIBLIOGRAPHY
61
LIST OF FIGURES
Figures
1. CNTF and CNTF receptor complex
Page
5
2. Three dimensional model of human CNTF
19
3. Jak-STAT signal transduction pathway
22
4. pSK-rCNTFR plasmid
35
5. Rat CNTFRa mRNA sequence
38
6. Effects of 12 hours of CNTF treatment on L8 cell protein synthesis
40
7. Effects of 24 hours of CNTF treatment on L8 cell protein synthesis
42
8. Effects of 24 hours of CNTF treatment on L8 cell total protein degradation 43
9. Radioactivites remaining in myofibrillar and non-myofibrillar proteins following
24 hours of CNTF treatment
45
10. Effects of 24 hour CNTF treatment on L8 cell total protein accretion
46
11. 35S-methionine autoradiography, myofibrillar proteins
49
12. 35S-methionine autoradiography, non-myofibrillar proteins
50
13. Northern blot of CNTFRa expression in L8 cells
51
14. Results of RT-PCR analysis of CNTFRa expression
52
DEDICATION
This thesis is dedicated to my family:
my parents, Ti-Chiou and I-Mei, for their endless love and trust
my brothers, Chih-Te and Chih-Ming, for their encouragement and support
which are so precious to my education and my life.
EFFECTS OF CILIARY NEUROTROPHIC FACTOR (CNTF) ON
PROTEIN TURNOVER IN CULTURED RAT L8 MUSCLE CELLS
INTRODUCTION
The neuromuscular system controls all movement of the animal body. Skeletal muscle
is highly regulated by nervous tissue both in excitation contraction and in supporting trophic
effects (McComas, 1996). Muscle atrophy, so called denervation atrophy, is observed after
muscle denervation and results from loss of neuronal stimulation and muscle metabolic
changes (Fernandez and Donoso, 1988). Several neurogenic substances are claimed to
encode trophic information affecting muscle cells. Therefore the goal of this research was to
investigate a putative neuron-derived myotrophic factor which regulates muscle protein
turnover.
Skeletal muscle is a high quality protein source for human consumption. About 40
percent of the body consists of skeletal muscle (Guyton, 1991). Muscle protein is classified
into two groups: myofibrillar proteins (55 percent of total muscle protein, organized into
contractile elements) and non-myofibrillar proteins (Allen, 1988). Muscle protein accretion
is regulated by both protein synthesis and protein degradation. For growing animals, the
synthesis rate is usually higher than the degradation rate (Samarel, 1991). An unbalanced
protein turnover rate always causes physical problems: muscle atrophy (degradation rate is
higher than re-synthesis rate) results in reduction in muscle mass and muscle hypertrophy (re­
synthesis rate higher than protein degradation rate) increases muscle mass (Guyton, 1992).
Several factors are known to cause muscle wasting or muscle atrophy: motor neuron
diseases or denervation as in amyotrophic lateral sclerosis (ALS, for review, see McComas,
2
1996; Williams and Windebank, 1991); immune cytokine events such as cachexia (Henderson
et al., 1994); genetic disorders such as Duchenne dystrophy (McComas, 1996); and
nutritional deficiencies such as White Muscle Disease (Shamberger, 1983). Each of these
situations presents a different causation; however, they each result in loss of muscle protein
via an increase in protein degradation. In this study, we focused on a neuron-derived
cytokine: ciliary neurotrophic factor (CNTF).
Ciliary neurotrophic factor (CNTF) is a cytokine which exerts a neurotrophic action.
It was first found in chick embryonic eye tissue in 1979 by Adler et al. It was recognized as
a putative neurotrophic factor because it supported differentiation or survival of different
kinds of neurons. These included sensory, sympathetic and motor neurons, the central
nervous system and the peripheral nervous system (Sendtner et al., 1994). Using in situ
hybridization methods, CNTF was found in astrocytes in the central nervous system and in
Swann cells in the peripheral nervous system (Stockli et al., 1991). Immunochemistry studies
showed that CNTF is largely released after denervation or motor neuron damage (for review,
see Sendter et al., 1994) suggesting that CNTF plays an important role in the function of the
neuromuscular system.
Based on about twenty published papers (for review, see Stahl and Yancopoulos,
1994) the CNTF receptor consists of a tripartite receptor complex. The CNTF specific
receptor, (CNTFRa), is the subunit which recognizes and binds directly to CNTF. Following
the combination of two other transmembrane receptors, LIMO (leukemia inhibitory factor
receptor 13) and gp130, the receptor complex activates the Jak-STAT signal transduction
pathway in response to CNTF (Figure 1). CNTFRa was found to be highly restricted in
3
neural tissue. Interestingly, CNTFRa is also expressed at a high level in skeletal muscle
(Davis et al., 1991). The muscle CNTFRa mRNA is largely increased after denervation
(Davis et al., 1993). This indicates a specific CNTF function in skeletal muscle cells,
suggesting that CNTF plays a role in adaptation of skeletal muscle to denervation.
Progress in understanding the effect of CNTF on muscle was made by Helgren's
group in 1994 (Helgren et al., 1994). Administration of CNTF to denervated muscle in vivo
significantly attenuated denervation atrophy by both morphological and functional
assessments. CNTF reduced muscle wasting after denervation in both fast and slow muscle.
This led us to question the mechanism by which CNTF exerted control of protein homeostasis
in skeletal muscle.
The goal of our research was to examine the mechanism by which CNTF exerts
trophic effects on skeletal muscle. In this study, we used cultured rat skeletal muscle cells
(L8 cell line) as a model. Our hypothesis was that CNTF exerts trophic effects on skeletal
muscle by both increasing muscle protein synthesis and reducing muscle protein degradation.
Following CNTF treatment, protein synthesis, protein degradation and total protein
accumulation were measured. The expression of CNTFRa mRNA in L8 cell line were
compared with CNTFRa mRNA expressed in rat skeletal muscle. Results from this study
may be used to:
1. Define the role of CNTF in denervation muscle atrophy.
2. Suggest applications for CNTF in clinical neuromuscular disorder patients or veterinary
therapy for inflammatory or stress causing immune cytokine activation.
3. Clarify cytokine's regulation of skeletal muscle metabolism.
4
4. Investigate potential use of CNTF as a strategy to enhance muscle growth or efficiency
of muscle growth.
5
CNTF
LIFR-f1
/130
Cell membrane
STATs
Figure 1. CNTF and CNTF receptor complex. After CNTF binds with CNTFRa, the signal
transduction machinery is activated via the Jak-STAT signal transduction cascade (for review,
see Stahl and Yancopoulos, 1994).
6
LITERATURE REVIEW
Muscle and Neuron
( 1 ) Skeletal Muscle and Muscle Proteins
Skeletal muscle is the best known and understood muscle of the four types of muscle
tissue. In addition to its role in movement, it is also the major amino acid pool in starvation
and heat production in the animal body (Ferguson, 1985). Sarcomeres, the unit of muscle
physiological function, comprise the major mass in muscle and permit contraction for force
generation and transmission (Bates et al. 1983). The individual proteins of the myofibrils are
continuously undergoing degradation and synthesis. Both of these processes are dependent
on the age of the animal, the subtype of the muscle, activity and health and endocrine states
(Bates et al., 1983, Allen 1988; Millward, 1980).
The skeletal muscle cell, or muscle fiber, is a multi-nucleated cell which originates
from the embryonic mesoderm. Although muscle cells retain the major organelles present in
most cells, the unique subcellular aspect of muscle fibers is the contractile machinery, the
myofibrils. Twelve to fourteen proteins organize the contractile unit (Allen, 1988) and are
classified as "myofibrillar proteins". These specialized proteins comprise about 55% of the
total protein in muscle cells. The rest of the protein is classified as non-myofibrillar protein
and includes cell membrane proteins, enzymes, nuclear proteins, transcription factors, and so
on. Myofibrillar proteins are insoluble in low ionic strength buffers.
The myofibril consists of two major filaments (a-actin and myosin heavy chain)
overlaid in a hexagonal array which creates the basic unit of the striated structure (Allen,
7
1988; Ferguson 1985; Voet et al., 1995). The thick filament is composed of myosin, a protein
consisting of many molecules laid parallel to one another to form a single, thick filament
which is about 15 nm by 1500 nm. The major component in the thin filament is aactin,
which is roughly 6 nm by 1000 nm. Within the fibril, the individual contractile unit is the
sarcomere. This can be longitudinally defined as the space between two Z lines which anchor
the actin filaments. The sarcomere can be divided into an I-band, which is the region of a
actin only; the A-band, corresponding to the position of the myosin filaments, and the H-band
at the center of the sacromere where actin and myosin do not overlap. According to the
hypothetical scheme of cross-bridge cycling (Rayment et al, 1993, 1994), the hydrolysis of
ATP at the globular head of the myosin (which is referred as Si fragment) initiates the power
stroke, followed by the myosin head "walking" up the actin thin filament. The sarcomere
shortens and the contractile cycle lasts approximately 50 ms.
The troponin complex (three polypeptides of troponin I, troponin C, troponin T) and
tropomyosin are associated with the actin fibril and regulate the contractile machinery. In
response to a rise in the concentration of CC, troponin C binds to CC and undergoes a
conformational change to lift the tropomyosin from actin so that the myosin heads becomes
attached, allowing the contraction to proceed (Guyton, 1991; McComas, 1996). Two giant
proteins, titin (also known as connectin) and nebulin were discovered to be "protein rulers"
which regulate the assembly of myosin and a actin filaments precisely and to maintain the
highly ordered structure of muscle both in elasticity and stability (Trinick 1994; Labeit et al.,
1995). All the myofibrillar proteins contribute in the beautifully ordered structures.
8
Non-myofibrillar proteins can be dissolved in low salt buffer whereas the myofibrillar
proteins are soluble in high salt buffers. This difference in solubility is used as a method of
separating the myofibrillar and non-myofibrillar proteins (Go 11 et al., 1989).
The non-myofibrillar proteins function in control of muscle growth, metabolic
regulation, and signal transduction.
Examples of non-myofibrillar proteins with key
regulatory features include glucose transporters (Zierath 1995); membrane bound proteins,
which serve as ion channels or pumps; and receptors, which function in response to either a
change in the transmembrane voltage or to signals from the neuromuscular junction or from
a blood transmitter (Guyton, 1991). Cytocellular proteins, such as proteins involved in signal
transduction and proteins which control muscle differentiation, all play important roles in
controlling muscle cell function and are included in the non-myofibrillar fraction.
( 2 ) Protein Degradation
Two decades ago, researchers discovered that not only the soluble cellular proteins
(i.e., non-myofibrillar proteins) turn over with widely differing half-lives, but the myofibrillar
proteins are also continuously undergoing degradation and replacement (Millward, 1980).
Most of the myofibrillar proteins turnover at non-uniform rates and have a long half-life, up
to several days (Low et al., 1973; Koizumi, 1974; Goll et al., 1989). The soluble proteins in
muscle have half-lives ranging from less than 30 mins to over 200 hours (Creighton, 1993)
with similar turnover characteristics to those observed in other tissues (Millward, 1980).
In the past ten years, considerable progress has been made in identifying mechanisms
which account for the degradation of muscle proteins. It is now known that at least four
9
proteolytic systems are involved in degradation of the intracellular skeletal muscle proteins.
Theses systems include:
1. Lysosomal proteases (cathepsins)
Lysosomal proteases play an important role in degradation of endocytosed proteins
and many membrane proteins by both selective and non-selective pathways (Dice, 1990;
Creighton, 1993). The known myofibrillar substrates for lysosomal proteases, based on in
vitro studies, include myosin heavy and light chains, a-actin, troponin, and desmin in vitro
studies (Okitani et al., 1988; Whipple et al., 1991). However, this is not the major pathway
for complete skeletal muscle myofibrillar protein breakdown in vivo (Lowell et al., 1986).
The acid lysosomal proteases, comprising the aspartic protease cathepsin D and the cysteine
proteases, cathepsins B, H, L and S (Ouali et al., 1993), were found to make specific
contributions in myopathic protein breakdown (Hall et al., 1996; Gogos et al., 1996). Their
role in myofibrillar protein degradation or the contribution in the process is probably limited.
Goll et al. (1989) reported limitations of the lysosomal cathepsins in degrading myofibrillar
proteins. These included the inability of the lysosome to engulf intact myofibrillar proteins
and the low pH-optima of the lysosomal cathepsin. It is now believed that the lysosomes, at
most, may participate only in the terminal degradation of myofibrillar fragments to produce
free amino acids (Solomon et al., 1996).
10
2. Ca' dependent proteases (calpains)
The cytosolic cysteine proteases consist of at least 6 isoenzymes including u-calpain,
m-calpain, p94,
calpain, and two stomach-specific calpains (Sorimachi et al., 1995, 1994;
Saido et al., 1994). A typical subset of cellular proteins has been shown to be calpains
substrates. These include proteins which participate in cell signal transduction, cytoskeletal
proteins, membrane receptors, calmodulin-binding proteins, transcription factors, and
enzymes (Wang et al., 1994; Saido et al., 1994). With reference to myofibrillar protein
degradation, calpains were first found to be responsible for the Z-disk degradation which is
associated with in the rapid structural alteration of myofibrils (Busch,
1972).
Immunohistochemical studies suggest that calpains play an important role in muscular
dystrophy (Kumamoto et al., 1995). However, undenatured myofibrillar proteins incubated
with purified calpain indicated that calpains cannot degrade the myofibrillar proteins to small
peptides or amino acids (Go 11 et al., 1992). This indicates that calpain may disassemble the
ultrastructure of muscle fiber, for example, by releasing a-actinin/a-actin from Z-disk (Goll
et al., 1991), and/or by making specific cleavages that release thick and thin filaments from
the sarcomeric ultrastructure and dissociate the large polypeptide fragments from other
myofibrillar proteins (Goll et al., 1992).
3. Proteasome, multicatalytic proteinase complex (MCP)
Proteasome (2000 kDa) is a large protein complex containing a 20S (700 kDa)
cylinder-shaped proteolytic core which contains multiple peptidase activities, and a 19S
regulatory complex composed of multiple ATPases and components necessary for binding
11
protein substrates (Peters 1994; Coux et al., 1996; Tamura et al., 1995; Goldberg 1995).
Ubiquitin conjugation with the protein target is believed to be the rate-limiting step in the
ATP-ubiquitin-proteasome pathway (Ciechanover 1994; Solomon 1996). A variety of
proteins were shown to be substrates of the proteasome including rate-limiting enzymes,
transcription factors, abnormal proteins, and even long-lived normal proteins (Argiles et al.,
1996; Rock et al., 1994). In addition, proteasome was found to be active in several
pathological states such as cell suicide (apoptosis), cancer cachexia, infection and injury,
starvation and malnutrition, muscle denervation atrophy, and metabolic acidosis (Argiles et
al., 1996; Furuno et al., 1990; Shinohara et al., 1996; Wing et al., 1995; Temparis et al.,
1994). ATP also was found to stimulate skeletal muscle endogenous protein breakdown up
to 12-fold (Fagan et al., 1987). In muscle tissue, myofibrillar proteins, actin, myosin, desmin,
and so on, were digested in an ATP-ubiquitin pathway (Taylor et al., 1995) into short
polypeptides but were protected when they associated as actomyosin complex or in intact
myofibrils (Solomon et al., 1996). It has been concluded that the proteasome play a key role
in degrading released myofibrillar proteins. However, there is no evidence that this
proteolytic structure is able to disassemble the intact myofibrillar complex.
4. Metalloendoproteases
Metalloendoproteases can be activated at the micromolar levels of Zn", Co", or Mn"
and have different cellular locations and physiological functions. For example, the insulin-
degrading enzyme (IDE, a cytosolic metalloendoprotease) plays a role in developmental
regulation for it can degrade insulin and insulin-like growth factors (Kuo et al., 1993). The
12
matrix metalloendoproteases were reported to be involved in the degradation of connective
tissue and have been implicated in the control of morphogenesis (Goo ley et al., 1993; Olsen,
1996). Although metalloendoproteases are also found in muscle tissue (Kirshner et al., 1983),
their function in this tissue is not well known.
( 3 ) Protein Synthesis
As mentioned previously, the proteins of the myofibrils exist in a dynamic state
(Millward, 1980). It is important that these proteins do turnover because muscle represents
an important source of amino acids and energy during diseases, stress and starvation. Muscle
protein accretion or accumulation is dependent on both the rate of muscle protein synthesis
and the rate of the muscle protein degradation (Schoenheimer et al., 1940). Synthesis rate
exceeds degradation in a growing animal but the degradation rate is larger than synthesis in
muscle wasting (Allen, 1988). The rate of protein accumulation varies according to muscle
fiber type (Millward, 1980), age, feeding or starvation situation, disease or hormonal status
(Bates et al., 1983; Sugden et al., 1991; Allen, 1988).
Protein synthesis can be measured both in vitro and in vivo by measuring the
incorporation of isotopically-labeled amino acids into protein (Samarel, 1991). In 1983, Bates
et al. found that the synthesis rate of myofibrillar proteins was more sensitive to nutritional
state than was the synthesis of intracellular soluble proteins.
Several factors have been
reported to affect myofibrillar protein synthesis in muscle development.
summarized in the following table:
These are
13
example
Factors
Hormones
small compounds
references
Insulin
Goll et al., 1989
Growth factors: IGFs
Hong et al., 1994
Steroid hormones
Allen, 1988
amino acids: glutamine
Wu et al., 1990
MacLennan et al., 1987
Glucose
Fulks et al., 1975
2nd messenger: cAMP
Sugden et al., 1993
physical factors
stretch
Perrone et al., 1995
Immune factors
Cytokines; IL-15
Quinn et al., 1995
In this study we focused on the role of CNTF, a recently-discovered neurotrophic
factor, which has been reported to exert trophic effect on skeletal muscle.
( 4 ) Neuron and Muscle
It has been known for a long time that muscle is excited by motor neural stimulation.
Cross-innervation experiments indicated that the nerves determine phenotypic characteristics
of muscle fibers (Romanul et al., 1967; Dubowitz, 1967). The late stage of muscle
development was reported as being neurally dependent (Martin et al., 1993; McLennan,
1994). Neurons also exert trophic effects on muscle not only by imposing patterns of
contractile activity but also by provision of neurogenic tropic substances (MacComas, 1996;
MacLennan, 1994).
14
Denervated muscle is a good model to investigate the effects of neurogenic agents on
muscle. Changes in denervated skeletal muscle result both from interruption of electrical
activity and loss of neurogenic trophic substances (Davis et al., 1980; Valenzuela et al.,
1995). Muscle denervation atrophy leads to a rapid reduction in cross-section areas of fibers
(i.e. wasting), protein content and contractile strength and is accompanied with ultrastructural
changes (Engel et al., 1974; Heck and Davis, 1988). Overall protein breakdown was
increased 3-fold in rat soleus muscle after 3 days of denervation, and changes in the multiple
proteolytic systems of muscle paralleled the denervation atrophy (Furuno et al., 1990). Non­
lysosomal ATP-dependent proteolysis was reported to be the main degradative route for
myofibrillar components (Wing et al., 1995). This finding was confirmed at the mRNA level.
In denervation atrophying muscle, mRNA for several proteasome subunits increased two to
four fold but no change in mRNAs encoding lysosomal enzymes and calpain (Medina et al.,
1995). This process is an important process in many neurological diseases.
Based on these findings, neurogenic effects in muscle may be investigated by a reverse
strategy: administration of nerve extract, supply of neurogenic factors, or re-innervation.
Administration of nerve extract ameliorates the denervation atrophy by reducing the loss of
weight, protein, and cross-sectional area of the fiber (Heck and Davis, 1988). Several
neurotropins were found to affect muscle development or survival. For instance, sciatin
(transferrin), found in sciatic nerve, maintains the maturation and survival of muscle cells
(Markelonis et al., 1979). As far as it is known, all the effects of denervation can be reversed
by re-innervation, including the maintenance of the specialized neuromuscular-muscle
15
junction, metabolism, membrane characteristics, and muscle contractile behavior (Mac Comas,
1996; Gutman, 1976).
In addition, muscle also produces trophic factors which support motoneurons. This
phenomenon is referred to as "nerve-muscle cell trophic communication" (Fernandez et al.,
1987). It is well-accepted that the death of neurons during development results from the
failure of muscle to gain the sustaining trophic molecules from their target tissue (Oppenheim,
1989). In an in vivo study, muscle extracts promoted motor neuronal survival in a dose-
dependent fashion (Oppenheim et al., 1993). This agrees with an in vitro study (BlochGallego et al., 1991). Similar studies didn't show the trophic effects in the presence of tissue
extracts from liver, lung, or heart (Oppenheim, 1989).
Further research with avian
motoneurons showed that the skeletal muscle-derived trophic factors prevented motoneuron
programmed cell death (apoptosis; Comella et al., 1994). Muscle extracts also showed
functional support of motor neurons. The production of neurotrophin-4 (NT-4) in rat skeletal
muscle was found to act as a neurotrophic signal for growth and remodeling of adult motor
neuron innervation (Funakoshi et al., 1995). In conclusion, the relationship between neuron
and muscle is highly regulated and inter-dependent. Here we investigate one aspect of this
relationships: the effects of CNTF on muscle protein homeostasis.
Ciliary Neurotrophic Factor (CNTF)
(1) CNTF
Ciliary neurotrophic factor was first characterized as a survival factor for ciliary
neurons (Adler, 1979). The chick ciliary neuron was chosen for study of the neuron muscle
16
junction because it could be easily isolated (Hooisma et al., 1975). It was found to survive
when co-cultured with chick skeletal muscle (Betz, 1976; Nishi et al., 1977), eye tissue
(Ebendal et al., 1980) or sciatic nerve extract (Richardson et al., 1982). The putative survival
factor in those tissues was later identified as an essential survival factor for the 8-day chick
embryo ciliary neuron. It was found to be rich particularly in the iris, ciliary body, and
chorioid layer (Adler et al., 1979; Manthorpe et al., 1982). Finally, CNTF was purified from
chick eye by Barbin et al. in 1984.
The CNTF cDNA sequence was first published in 1989 by Lin et al. (rabbit) and
Stock li et al. (rat). These two cDNAs were found to have 80% identity (Masiakowski et al.,
1991). The cDNA-deduced amino acid sequences indicated that CNTF is a cytosolic protein.
Using the cDNA and a genomic DNA fragment including the entire intron region as mixed
probes, the CNTF gene was located to the long arm of human chromosome 11 by in situ
hybridization (Yokoji, et al., 1995) and rat chromosome 19 (Kaupman et al., 1991).
The CNTF mRNA and protein are synthesized primarily by glial cells and Schwann
cells in the peripheral nervous system (PNS) and by astrocytes in the central nervous system
(CNS; Stolckli et al., 1991). Although CNTF was first identified as an essential factor in
embryonic culture, its developmental role in vivo is not well understood. By Northern blot
analysis, CNTF mRNA did not appear in rat embryonic brain until 11-days of development
(Ip et al., 1993). CNTF mRNA was expressed in rat as early as postnatal day 5 by in situ
hybridization measurements (Seniuk-Tatton et al., 1995). In adult animals, the highest levels
of CNTF mRNA were detected in peripheral nerves such as sciatic nerve (Williams et al.,
1984) and in the RNA from optic nerve, olfactory bulb and spinal cord in the central nervous
17
system (CNS; Stolckli et al., 1991; Ip et al., 1993). CNTF mRNA could not be detected in
spleen or lung (Stock li et al., 1989)., and only a very weak signal was detected in skeletal
muscle (Ip et al., 1993). By highly sensitive in situ hybridization methods, CNTF mRNA was
detectable in nuclei of muscle fiber cells (Seniuk-Tatton et al., 1995).
The molecular cloning of CNTF cDNA showed that the protein consisted of 200
amino acids with a calculated molecular weight of 22.8 kDa and 24 kDa for rat and human,
respectively (Negro et al., 1991; Masiakowski et al., 1991). The acidic protein (pI about 5.8)
(Negro et al., 1991), is highly conserved across species as the protein sequences of human
and rat have 83% identity (Lam et al., 1991). From circular dichroism measurements, the
CNTF protein is a polypeptide which contains four anti-parallel a-helices (A, B, C, D
respectively; Bazan, 1991; Kruttgen et al., 1995). It shares sequence homology with
interleukin 6 (IL -6), leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1
(CT-1) and interleukin 11 which, together, are classified as the IL-6-type cytokine family (for
review, see Shields et al., 1995). In this family, all the members share the common signal
transduction receptor in addition to their own specific receptor (for review, see Stahl and
Yancopoulos, 1994). Because of CNTF's specific neurogenic characteristic, CNTF was also
classified as a "neuroproietic cytokine" or "neurocytokine" (for review, see Taga, 1996).
Although CNTF shares a highly homologous sequence with other IL-6 family
members, CNTF lacks a hydrophobic leader sequence usually found in proteins which are
secreted by the classical endoplasmatic reticulum-Golgi pathway (Lin et al., 1989). Structure-
function information about CNTF was obtained by site-directed mutagenesis. Both N-(14
mer) and C-(18 mer) terminus truncations retain biological activity (Kruttgen et al., 1995).
18
The replacement of LYS-155 of hrCNTF (human recombinant CNTF) with any other amino
acid residue resulted in abolishment of neural cell survival activity, and Glu-153 mutants had
5- to 10- fold higher biological activity. The two amino acids located in the Dl structure
motif (the beginning of the D-helix of CNTF protein, Figure 2) play a key role in the receptor-
signal transduction pathway (Inoue et al., 1995; Di-Marco et al., 1996). The amino acid side
chains in helix A, AB loop, helix B and D are important in CNTF-specific receptor
recognization (Panayotatos et al., 1995; Bazan 1991).
(2) CNTF Rreceptor Components
Research has identified a three-component receptor complex for CNTF and its signal
transducing pathway. Citing from more than 15 papers (for review, see Stahl and
Yancopoulos, 1994) the binding of CNTF to the CNTF-specific component, known as
CNTFRa, produces most of its effects and promotes the binding to the signal transducing
components, gp130 and LIFRI3. The function of the CNTFRct subunit is to increase CNTF
affinity for the transducing receptor components. These two transmembrane heterodimer
components are pre-associated with Jak/Tyk kinases. Following CNTFRa binding to the two
proteins, the combination initiates a series of intracellular tyrosine phosphorylation that
ultimately lead to changes in gene expression through the Jak-STAT signal transducing
pathway (Figure 1).
The human CNTFRa gene is located on chromosome 9 (Valenzuela et al., 1995).
Similar to the high conserved CNTF protein sequence, rat CNTFRct is 94% identical to its
human counterpart (Ip et al., 1993). CNTFRa shares homology with its family member, IL-6
19
Figure 2. Three dimensional model of human CNTF. Predicted 4 helices were marked as A,
B, C, and D. The proposed receptor binding site (D1 cap region) is circled. (The figure was
modified from Kruttgen et al., 1995).
20
receptor a (IL-6a; for review, see Stahl and Yancopoulos, 1994). Members of this family
contain a conserved domain in their receptor extracellular region by positional conserved
cysteine residues and a WSXWS motif near the carboxy-terminal end (Bazan, 1990).
CNTFRa is a hydrophilic acidic protein with the molecular weight of about 41kDa
(372 amino acids) and has a pI of 6.5 based on its cDNA sequence (Davis et al., 1991;
Panayotatos et al., 1994). The receptor-ligand complex binds with 1:1 stoichiometry. Unlike
other growth factor receptors, CNTFRa is anchored to the cell membrane by a glycosyl­
pho sphatidylinositol (GPI) linkage (Davis et al., 1991). Like other GPI-linked proteins,
CNTFRa can be released from the cell membrane by phosphatidylinositol-specific
phospholipase C (PI-PLC; Davis et al., 1991). Released soluble CNTFRa (sCNTFRa) has
a potential physiological role in mediating CNTF responses (Davis et al., 1993).
As would be expected, CNTFRa expression is largely limited to the known targets
of CNTF action, mainly in the nervous system in adult animals (Davis et al., 1991; Ip et al.,
1993; MacLennan et al., 1996). Interestingly, CNTFRa is also expressed at high levels in
skeletal muscle (Davis et al., 1991; 1993). Furthermore, CNTFRcc was detected widely in
embryonic nervous tissue and in postnatal nervous system, muscle, and liver (Kirsch and
Hofmann, 1994; MacLennan et al., 1996; Ip et al., 1993). Mutant mice, lacking CNTFRa,
die perinatally and display severe motor neuron deficits (DeChiara et al., 1995). Although
CNTF expression is barely to detected in the embryo, embryonic expression of CNTFRa
indicates that CNTF plays an important role during development.
21
Because it lacks a cytoplasmic domain, CNTFRa must interact with other membrane-
bound components to initiate biological action. Two membrane proteins, gp130 and LJkRJ3,
are known to associate with CNTFRa. These proteins are members of a distant protein
family, including CNTF, IL-6, LIF, OSM, and IL-11 (for review, see Shields et al., 1995;
Taga, 1996; Stahl et al., 1994). This related cytokine family initiates signaling by binding the
ligand with the a component to induce either homodimerization of the gp
receptor
components (such as IL -6) or heterodimerization between gp130 and LIFR (such as CNTF;
Davis et al., 1993).
(3) Signal Transduction: Jak-Stat Pathway
LIFR13 and gp 130 are closely-related, having similar transmembrane and cytoplasmic
regions (Gearing et al., 1991). The cytoplasmic region contains 3 modular tyrosine-based
motifs which can be phosphorylated in the signal transduction cascade. Two conserved
membrane proximal domains of the receptor components are preassociated with Jaks (Janus)
kinase (Stahl et al., 1995). So far, there are four members in the jak family known: Trk 2, Jak
1, Jak 2, and Jak 3, with molecular weights ranging from 125-135 kDa (for review, see
Schindler and Darnell, 1995; Darnell, 1996). Two tandem tyrosine kinase domains of the Jaks
are highly-conserved across the family members and are believed to rapidly activate by a
ligand-stimulated phosphorylation (Darnell, 1996).
Stahl et al described a phosphorylation cascade model in 1995 (Figure 3). The binding
of CNTF to its a components triggers the dimerization of the two transmembrane
22
Dim erization and
nuclear translocation
Figure 3. Jak-STAT signal transduction pathway. Signal transduction (ST) receptor
components were pre-associated with Jak proteins. The binding of CNTF and CNTFItot
induces hetrerodimerization of the ST components then activates the signal transducing
cascade (for review, see Ip et al., 1996).
23
components and the associated Jak kinase, then results in tyrosine phosphorylation and
activation of the Jaks, which then phosphorylate the receptor's tyrosine-based motifs. The
phosphorylated motifs form a binding site for the STATs (signal transducer and activators of
transcription) protein family .
STATs are named for the dual function in signal transduction in the cytoplasm and
activation of transcription in the nucleus (for review, see Horvath and Darnell, 1997). The
STAT family is composed of 6 stat proteins: STAT 1 to 6. STAT 3, also known as acute-
phase response factor, has been reported to be activated in response to the IL -6 cytokine
family in central nervous system primary culture (Rajan et al., 1996).
STATs contain
approximately 800 amino acid residues with a DNA binding domain at residues 400-500
(Horvath et al., 1995). The receptor-kinase complex interacts with a highly-conserved Src
homology (SH) 2 domain (Heim et al., 1995). N-termini of the STATs mediate several
protein-protein interactions or cooperative binding to tandem DNA (Xu et al., 1996).
According to the model of Stahl et al (1995), after binding with the receptor tyrosine-
based motifs, STATs become tyrosine phosphorylated, subsequently dissociate from the
receptors, and dimerize as either homodimers or heterdimers of STATs (in which the
phosphotyrosine of one partner binds to the SH2 domain of the other), and then translocate
to the nucleus. Several DNA sequence elements has been found that are recognized by STAT
proteins with a GAS (IFN-gamma activation site) consensus sequences (Schindler and
Darnell, 1995). Additionally, it was reported that the genes triggered by CNTF binding are
some of the early response genes such as jun-b, Its] I and c-fos (Wang and Fuller, 1995;
Bonni et al., 1993).
24
(4) Functions of CNTF
In vitro, CNTF promotes survival and/or differentiation of several kinds of neurons
including parasympathetic, sympathetic, sensory, and motor neurons(for review, see Sendtner
et al., 1994; Richardson, 1994). As in embryonic culture, CNTF is necessary for E8 ciliary
(parasympathetic) ganglia neuron survival, and for E 1 0 -11 chick sympathetic and sensory
neurons (Manthorpe et al., 1982). More than 60% of E6 motoneurons survive in the
presence of CNTF and the survival is enhanced by FGF (Arakawa et al., 1990). However,
sympathetic neurons also undergo into apoptosis at high doses of CNTF (Kessler et al.,
1993). This indicates that CNTF could regulate more than one cellular event and that its
action could be dose-dependent.
CNTF advances neuronal development. In cultured neurons from E5 chick embryos,
CNTF didn't increase survival rate but promoted fiber outgrowth (Bianchi and Cohan, 1993).
CNTF also increases ChAT (choline acetyltransferase) activity in sympathetic neuronal
cultures (Saadat et al., 1989). CNTF also up-regulates expression of vasointestinal peptide
(VIP) and substance P (SP) which are ACh associated peptides (Ernsberger et al., 1989; Rao
et al., 1992). This demonstrates that CNTF induces cholinergic properties in adrenergic
sympathetic motor neurons. The regulatory action of CNTF on CNS neurons remains largely
unknown, but may include enhanced expression of NGF receptor (Magal et al., 1991) and
tyrosine hydroxylase (Louis et al., 1993). Taken together, these in vitro studies showed that
CNTF affects neurons both morphologically and functionally.
In vivo, CNTF also exerts multiple effects on survival and function. When exogenous
CNTF was added to developing chick embryos between E5 and E9, a significant increase of
25
the number of spinal motor neurons was observed (Oppenheim, 1991). In adult animal
studies, prevention of motoneuron degeneration by exogenous CNTF was reported in several
mouse models of motoneuron disease (Sendtner et al., 1992; Mitsumoto et al., 1994). Similar
studies with CNS neurons have shown that CNTF prevents both degeneration (Hagg et al.,
1992) and axotomy-induced cell death (Clatterbuck et al., 1993). Disruption of the CNTF
gene by homologous recombination results in a progressive atrophy and loss of motor
neurons in adult mice (Masu et al., 1993). However, CNTF has a profound effect on in vivo
embryonic development. CNTF mRNA is expressed at a low level in the embryo (Ip et al.,
1993). Abolishing endogenous CNTF expression produces mice which appear normal at birth
(Masu et al., 1993), while mice lacking CNTFRa die perinatally and display severe motor
neuron deficits (DeChiara et al., 1995).
Because CNTF immunoreactive cells are located widely in CNS and PNS, it is not
surprising that CNTF exerts its effects not only in neural cells but also in neuronal-related
cells, such as oligodendrocyte, astrocytes, Schwalm cells and microglial cells (for review, see
Sendtner et al., 1994). However, the spectrum of CNTF-responsive cells includes somatic
cells, even though CNTF protein or CNTFRa are not detectable in those tissues. Examples
include embryonic stem cells (Conver et al., 1993), hepatocytes (Schooltink et al., 1992),
adipose tissue (Nonogaki et al., 1996), and bone marrow (Bellido et al., 1996). Additionally,
CNTF is reported to induce cachexia in rodents (Henderson et al., 1994) and fever (Shapiro
et al., 1993) following systemic administration. These findings indicate that CNTF has
multiple biological activities.
26
Skeletal muscle is the only somatic tissue which expresses a high level of CNTFRct
(Davis et al., 1991). Several studies have addressed this specific expression, but so far, the
CNTF function in muscle is still largely unknown. In developing rats, CNTF prevents
bulbocavernosus motoneuron degeneration and atrophy of the target muscle (Forger et al.,
1993). Further study by the same group found that CNTF intervenes in preventing not only
muscle fiber atrophy, but also degeneration involving the death of muscle fibers (Forger et al.,
1995). In 1994, Helgren et al. reported that CNTF exerts a trophic effect on denervated
muscle both in attenuating the loss of morphological and functional properties. CNTF is
capable of reducing muscle wasting after denervation in both slow (soleus) and fast (extensor
digitorum longus, EDL) muscle, but the mechanism by which these effects are mediated has
not been elucidated. Loss of cross-sectional area and the contractile ability of denervated
muscle was also reduced in CNTF-treated animals. CNTF has been applied in amyotrophic
lateral sclerosis patients because of its trophic effects both on motoneuron and skeletal muscle
(ALS CNTF treatment study group, 1996).
Controversy has developed regarding CNTF's trophic role. Recently, it was reported
that CNTF induced potent cachectic effects and caused rapid catabolism of adipose tissue and
skeletal muscle (Henderson et al., 1996). Martin et al. (1996) reported that hrCNTF (human
recombinant CNTF) caused atrophy of skeletal muscle based on the results of an in vivo
study. The muscle-wasting effects of hrCNTF were associated with the loss of body weight
and reduction in food intake. During the weight lost of CNTF-induced cachexia, acute phase
proteins were synthesized and both fat and muscle contents were reduced. These results were
similar to the cachexia induced by tumor necrosis factor (TNF), IL-6, and LIT (Henderson
27
et al., 1994). However, an in vitro study showed that the rate of total protein degradation and
protein synthesis were unchanged by co-incubating CNTF with rat EDL muscle (Espat et al.,
1996). The physiological role of CNTF in muscle protein turnover remains unclear.
Profound effects of CNTFRa mRNA expression have also been shown in denervated
muscle.
Davis et al. (1991) found that CNTFRa mRNA concentration was increased
following denervation of rat gastrocnemius. Confirming results were published by Helgren
et al. (1994) in EDL and soleus muscle. On the other hand, the down regulated CNTFRa
mRNA expression in denervated muscle was observed by Ip et al. in rat leg muscle (Ip et al.,
1995) and in chick (Ip et al., 1996). Since the CNTFRa is essential for CNTF function, the
regulatory mechanism of CNTF effects on muscle is unknown.
Regarding CNTF's family members, cytokines in the IL-6 family have been shown to
exert different effects on muscle tissue. IL-6 is reported to augment muscle proteolysis, and
may have a role in muscle breakdown during infection (Goodman, 1994). This accelerated
proteolysis results from changing both the lysosomal cathepsin pathway and ATP-ubiquintin
pathway (Tsujinaka et al., 1996). An in vitro study has shown that LIF promotes myoblast
differentiation (Vakakis et al., 1995). Both LIF and IL-6 were up-regulated following
denervation (Kurek et al., 1996) and may play an important role in muscle regeneration
(Kurek et al., 1996).
In this study, we used cultured rat muscle cells to study the mechanism by which
CNTF regulates protein homeostasis in skeletal muscle. Our hypotheses were that CNTF
exerts trophic effect in this tissue via control of both protein synthesis and degradation. To
28
gain insight into the mechanism by which CNTF affected control of protein turnover, we
assayed the expression and control of CNTFRct mRNA expression.
29
MATERIALS AND METHODS
Cell Culture
The rat myoblast L8 cell line was obtained from American Type Culture Collection
(ATCC; Rockville, MD) and was stored at -80 ° C in 10% dimethyl sulfcodde (DMSO) until
use. Working cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 100
units/ml of penicillin, 100 1.1,g/m1 of streptomycin (DMEM, penicillin and streptomycin, all
from Gibco, Grand Island, NY) and 44mM NaHCO3. The media was supplemented with 10
percent fetal bovine serum (FBS, Hyclone, Logan, UT) during the proliferation phase and
with 2% horse serum (Hyclone, Logan, UT) during the differentiation phase. L8 cells were
incubated in 5% CO2 and 95% air at 37°C. Microscopic examination was used to monitor
cell differentiation. The myotubes were used for experiments when they had reached 80-90%
differentiation.
Total Protein Accretion Measurement
Equal amounts of L8 cells were seeded onto 60 mm culture dishes (Corning, NY).
After reaching 80-90 % differentiation, cells were treated with CNTF in differentiation
medium. After CNTF treatment (see below), cells were washed with PBS and lysed with
alkaline lysis buffer (0.5N NaOH, 1% Triton X-100). A small portion of cell lysate was taken
for determination of protein concentration using the Bio-Rad (Hercules, CA) protein assay
reagent according to the method of Bradford, (1976). Cell lysate volume from each plate was
estimated by weight. The total protein amount was determined as protein concentration
30
multiplied by lysate volume. Total protein was then compared between the treatment (CNTF)
and control groups.
Separation of Myofibril lar Proteins and Non-myofibrillar Proteins
L8 cells were cultured in 60 mm culture plates. Cells were washed twice with ice-cold
PBS (phosphate buffered saline; pH 7.4), then scraped in 0.5 ml homogenizing buffer
containing 40 mM NaC1, 0.1mM EGTA, 1 mM dithiothreitol, 0.1% Triton X-100 in 5mM
sodium phosphate, pH 6.8.
Lysates were processed with 15 strokes of a Dounce
homogenizer and then centrifuged at 1000Xg for 5 min. The supernatant was saved as the
non-myofibrillar protein-rich fraction. The pellet, containing the myofibrillar fraction, was
dissolved in 0.5N NaOH with 0.1% Triton X-100.
Assessment of Protein Synthesis
Rates of protein synthesis were measured by monitoring the incorporation of 3H­
tyrosine into acid-insoluble material. Differentiated cells were treated with CNTF (0, 1, and
10 ng/ml) in differentiation medium. Two hours before the end of CNTF treatment, 3H­
tyrosine (ICN, Costa Mesa, CA) was added into CNTF medium to final concentration 1
p.Ci/ml. Cell lysate was harvested, myo- and non-myofibrillar proteins were separated, and
radioactivities associated with both fractions were determined by use of a scintillation counter
(LS6000 SE, Beckman, Pittsburg, PA). A small fraction was taken for protein analysis using
Bio-Rad (Hercules, CA) protein assay reagent. Protein content was assessed according to
31
Bradford (1976). Myo- and non-myofibrillar protein synthesis rates were calculated and then
compared with a blank control group.
Assessment of Protein Degradation
Differentiated cells were washed with DMEM (37 °C) twice then treated with 0.5
.tCi/ml of 3H- tyrosine in 2 mL DMEM-2% horse serum for 24hr. Ciliary neurotrophic factor
(CNTF) was purchased from R&D (Minneapolis, MN) and was dissolved in PBS. Following
tritium labeling, cells were washed with DMEM (37 °C) twice and re-supplemented with
differentiation media containing 2mM non-radioactive tyrosine and CNTF ranging from 0.5
to 20 ng/ml, then kept in an incubator for a designated time (12 or 24 hours). Following this
decay period, media samples were collected and trichloroacetic acid (TCA) was added to
media to a final concentration of 10% w/v. The sample was stored at 4°C for at least 1 hour
and then centrifuged for 10min, at 14000Xg. The supernatant was recovered and counted
in a liquid scintillation counter. Radioactivity in this fraction then served as an index of the
degradation of protein which had occurred. The total protein degradation rate was calculated
as the ratio of tritium released into media divided by the total medium CPMs + myofibrillar
fraction + non-myofibrillar fraction (see formula below).
To assess the specific degradation of the myofibrillar and non-myofibrillar fractions,
the cells in these treatments were harvested and separated into a myofibrillar fraction and a
non-myofibrillar fraction as described above. The radioactivities remaining in myo- and non­
myofibrillar proteins were determined by use of a scintillation counter. Protein degradation
32
rate was expressed as cpm remaining in each fraction divided by the total CPMs. Both myo­
and non-myofibrillar degradation were calculated by comparing with a blank control group.
Calculation Formula
Protein degradation rate (as a percent of the radiolabelled protein) was calculated as
the ratio of CPMs in the different fraction as follows
CPMs in the fraction of interest
Degradation =
Total CPMs (media + myofibrillar + non-myofibrillar)
The effects of CNTF on degradation and synthesis of various protein fraction were
expressed as percent of respective control.
35S- methionine Autoradiography Experiment
When L8 cells reached 90% confluence, cells were cultured in starvation media
(methionine/cysteine-free DMEM, from Gibco, with 2% HS) in a CO2 incubator for 30
minutes, then changed to labeling-media , 35S-methionine/cysteine; 20 uCi/m1 (ICN, Costa
Mesa, CA) in Met/Cys-free DMEM with 2% HS, and incubated for an additional 8 hours.
Cells were then washed with 37°C chase media (DMEM with 2% HS, 1 mM methionine and
1 mM cysteine), followed by incubation in chase media with CNTF for 24 hours. Myofibrillar
proteins and non-myofibrillar proteins were obtained according to the methods described
previously.
After separation, myofibrillar protein-rich fractions were dissolved in sample buffer
containing 2.3% SDS, 5% 2-mercaptoethanol and 6.25 mM Tris buffer, pH 6.8 in 10%
33
glycerol. Both myofibrillar protein-rich fractions and non-myofibrillar protein-rich fraction
were electrophoresized (50 pg each ) on a 7.5% to 15% gradient SDS-PAGE gel followed
by heat-drying of the gel under vacuum for 1 hour at 80 °C, then exposed to autoradiography
film (Reflection'', DuPont NEN, Boston, MA) for 24 hours.
Total RNA Extraction
Total RNA was extracted for Northern blotting based on the method of Chomczynski
and Sacchi (1987). Differentiated cells were treated with CNTF in differentiation medium.
After the treatment, the cells were washed with ice-cold PBS, pH 7.0, three times. The cells
were lysed in 0.5 mL solution A (containing 4M guanidium isothionate, 25 mM sodium
citrate, pH 7.0, 0.5% sarcosyl, and 10 mM (3-mercaptoethanol) or were stored at -80 °C until
use.
The cell lysate was scraped with a rubber policeman from the plate and transferred to
a microtube. Fifty 1AL of 2M sodium acetate, 0.5 nil. of phenol, pH 4, and 100 III. of
chloroform/isoamyl alcohol (49:1) were added into the tube. After addition, sample was
agitated and allowed to sit on ice for 20 minutes. The lysate was then separated by
centrifugation at 10,000 xg for 20 minutes, 4 °C. The upper aqueous phase was transferred
to a new tube and then an equal volume of ice-cold isopropanol was added. The sample was
mixed and frozen at -20 °C overnight. RNA was collected by centrifugation 10,000 xg for
20 minutes, 4°C. The supernatant was discarded and the RNA pellet was rewashed in 1 mL
of 75% ethanol, The RNA pellet was finally recovered by dissolving in 100 1.11, water and the
concentration was determinated by spectrophotometry.
34
CNTFRa cDNA Probe
CNTFR cDNA (Ip et al., 1993), subcloned in Bluescript pSK-rCNTFR (1.5kb-EcoRI,
Figure 4) phagemid, was obtained from Dr. Yancopoulos, Regeneron Pharaceuticals,
Tarrytowm, NY. This cDNA contained a partial CNTFRa fragment (1.5kb). A T3 promoter
was located at 5' end of the clone and T7 at the 3' end. The plasmid was transfected into
E. coli cells (DH5-a) by heat shock of the competent cells. E. coli were cultured in LB
medium and the plasmid DNA was extracted by alkaline lysis of the bacterial host cells
followed by polyethylene glycol (PEG) precipitation according to the method of Lis (1980).
The CNTFRa fragment was labeled with 32P-dCTP (DuPont NEN, Boston, MA) by PCR
(polymerase chain reaction) labeling with use of T3 and T7 primers (McDaniel et al., 1996).
A labeled cDNA fragment was purified by a Quick Spin G-50 Sephadex column (Boeringer
Mannheim, Indianapolias, IN).
a-Actin cDNA Probe
An a-actin northern blot was applied as an internal control. A rat muscle a-actin
partial cDNA sequence was cloned into pCRII plasmid by Dr. Gu (1997). The cDNA
plasmid was transfected into E. coli cells (Top 10F', Invitrogen, Carlsbad, CA) by heat shock
of the competent cells. The a-actin fragment (1130 bp) was obtained and PCR labeled as
described for the CNTFR cDNA probe. Specific primers to a-actin were applied in the PCR
labeling.
35
T3
EcoR1
pSK-CNTFR
4461 by
CNTFR
EcoRl
Figure 4. pSK-rCNTFR plasmid. The CNTFR cDNA is about 1.5 Kb (Ip et al., 1993).
36
Northern Blot Hybridization
RNA samples were denatured at 55 °C for 15 minutes, and applied to 1.1% agarose
gel containing 2.2M formaldehyde. Following gel electrophoresis at 30V for overnight, the
gel was washed three-times with DEPC-treated water. RNA was transferred to Nytran plus
membrane (Schlecher & Schuell, Keene, NH) and immobilized by ultraviolet multilinker
(UVC 515, Ultra-Lum inc., Carson, CA). The membrane was prehybridized at 42°C for 3
hours in prehybridization buffer (5x SSC, 50% formamide, 1% blocking reagent, N­
lauroylsarcosine 0.1%, 0.02% SDS).
Following prehybridization, the membrane was
hybridized overnight at 42°C. After washing three-times with 0.2x SSC and 0.1% SDS for
15 minutes at 42°, the RNA signal was detected by exposing the membrane to Kodak X-Omat
film (Kodak, Rochester, NY) with intensifying screens for 6-12 hours at -80 °C.
For re-probing, the membrane was washed with stripping solution (1% SDS, 0.1x
SSC, 40mM Tris -Cl) at 80 °C for 5 minutes. This was repeated three times prior to re-use
of the membrane.
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
The first strand cDNA synthesis was performed on 20 p.g total RNA. RNA was
heated to 70 °C for 5 minutes to melt secondary structure within the template. Reverse
transcription was carried out at 42 °C for 60 minutes in 20 1.d reaction solution containing 50
mM Tris-HC1, pH 8.3, 75mN KC1, 3mM MgC12, 10mM DTT, 10mM each dNTP, 0.5 m
oligo(dT)18 and 200 units of Moloney Murine Leukemia Virus Reverse Transcriptase (M­
MLVRT, Promega Corporation, Madison, MI). The reverse transcription reaction mixture
37
was diluted 1:6 with water and was used in polyreaction chain reaction. PCR reactions were
carried out in 50 !AL reaction solution containing 5 units of Taq DNA polymerase (Promega),
250 !AM of each dNTP, 2.5mM MgC12 and 3 p.M of CNTFR specific primers (primer 1 and
primer 3, figure 5). PCR was performed for 30 cycles at 94 °C for 2 min, 55 °C for 1 min and
72 °C for 1 min in a Techne PHC-2 thermal cycler (Techne, Princeton, NJ). Half of the
reaction was electrophoresed on 1 % (w/v) agarose gel and photos of gels were taken. To
identify whether the signal was from CNTFR mRNA, an internal primer (primer 2, Figure 5)
and primer 1 were chosen for a second (nested) PCR assay using at the conditions decribed.
Statistical Analysis
In the studies on the effects of CNTF on muscle cell proteins, each experiment was
repeated at least three times using three replicates for each treatment. Each treatment mean
(from 3 replicates) in the protein synthesis and protein degradation experiments was
compared with control group mean (no CNTF treatment). The ratio of CNTF treatment
group over control group was analyzed using multifactor ANOVA with a null hypothesis that
no difference exists between the CNTF treatment and control groups(ratio = 1). For total
protein accretion, the differences among different groups were assessed using multifactor
ANOVA. A level of significance of 5% was adopted for all comparisons and Statgraphic 7
(for Windows) was used as the statistical software.
38
1 caacacatca tcccaggaag actttggtct gcagaggagg ataatattga tgtgcttgga
61 gagcatctgg tggtaacgag ATGGCTGCTT CTGTCCCTTG GGCCTGCTGT GCTGTGCTTG
121 CCGCTGCCGC TGCCGCTGTC TACACCCAGA AACACAGTCC ACAGGAGGCA CCCCATGTTC
181 AGTATGAGCG TCTGGGCACA GATGTGACGC TGCCATGTGG GACAGCAAGT TGGGACGCAG
241 CTGTGACCTG GAGGGTAAAT GGAACAGATC TGGCCCCTGA CCTGCTCAAT GGCTCTCAGC
301 TGATACTACG AAGCTTAGAA CTGGGCCACA GTGGCCTGTA TGCCTGTTTC CACCGTGACT
361 CCTGGCACCT GCGCCACCAA GTCCTTCTGC ATGTGGGTTT GCCGCCTCGG GAACCCGTGC
421 TCAGCTGCCG 1TCCAACACTG TACCCCAAGG CTTCTACTG CAGCTGGCAC CTGTCCGCCC
Primer 1 (forward)
481 CCACCTACAT CCCCAATACC TTCAATGTGA CTGTACTGCA TGGCTCCAAA ATGATGGTCT
541 GTGAGAAGGA CCCAGCCCTC AAGAACCGCT GTCACATTCG GTACATGCAC CTGTTCTCAA
601 CCATCAAGTA CAAGGTCTCC ATAAGTGTCA GCAACGCCTT GGGTCACAAC ACCACGGCTA
Primer 2 (reverse)
661 TCACCTTCGA CGAATTCACC ATTGTGAAGC CCGATCCTCC AGAAAATGTG GTGGCCCGGC
721 CAGTGCCCAG CAACCCCCGT CGACTGGAGG TGACATGGCA GACCCCCTCA ACTTGGCCTG
781 ATCCCGAATC CTTTCCACTC AAGTTITITC TGCGCTACCG GCCTCTCATC CTGGATCAAT
Primer 3 (reverse)
841 GGCAGCATGT GGAGCTCTCG AATGGCACAG CCCACACCAT CACGGATGCC TATGCTGGGA
901 AGGAGTACAT CATCCAGGTG GCCGCCAAGG ACAATGAGAT TGGGACATGG AGTGACTGGA
961 GTGTGGCTGC TCACGCCACA CCCTGGACTG AGGAACCACG GCATCTCACC ACTGAAGCCC
1021 AGGCCCCCGA GACCACGACC AGCACCACCA GCTCCTTGGC ACCCCCACCC ACCACGAAGA
1081 TCTGTGACCC CGGGGAGCTC AGCAGCGGCG GAGGACCCTC CATACCCTTC TTGACCAGTG
1141 TCCCTGTCAC TCTGGTCCTG GCTGCCGCTG CTGCCACAGC CAACAATCTC ctgatctgag
1201 ccctgcatcc catgaggaca cgccagacgc ctacagagga gcaggaggcc ggagctgagc
1261 ctgcagaccc cggtttctat tttgcacacg ggcaggagga ccttttgcat tctcttcaga
1321 cacaatttgt ag
Figure 5. Rat CNTFRct mRNA sequence. Underlined regions were the primers which were
designed for RT-PCR reaction (sequence from Ip et al., 1993).
39
RESULTS
Effects of CNTF on L8 Mvotube Protein Synthesis
Rates of protein synthesis were measured by monitoring the incorporation of 3H­
tyrosine into cells. Rates of 3H-tyrosine incorporation into myofibrillar proteins and non­
myofibrillar proteins were reported to be linear during the first 30 hours after differentiation
(Ou, 1994). Based on this finding, the experiments shown here were limited to 24 hours in
duration. In the protein synthesis experiments, differentiated L8 cells were treated with
CNTF at varying doses (0, 1 or 10 ng/ml) for 12 and 24 hours. 3H-tyrosine was added into
the media during the last two hours of the treatments to minimize the effects of protein
degradation during the labeling. The final results were expressed as the ratio of 3H-tyrosine
incorporation rate between CNTF treatment and the control group (CNTF 0 ng/ml). A
multiple comparison procedure was applied to determine the difference between means with
significant level at p<0.05.
At 12 hours of CNTF treatment, both treated groups (CNTF dose 1 and 10 ng/ml)
were different from the control group. CNTF (CNTF dose 1, 10 ng/ml) increased myofibrillar
protein synthesis by 8.4 ± 2.7 % and 9.22 ± 3.18 % compared to the control group
respectively (Figure 6). CNTF did not significantly affect protein synthesis in the non­
myofibrillar fraction (Figure 6). The results showed that CNTF induced myofibrillar protein
synthesis after 12 hours of treatment.
40
Protein Synthesis, 12h
WM
Myofibril lar Protein
III.11 Non-myofibrillar Protein
10
1
CNTF, ng/ml
Figure 6. Effects of 12 hours of CNTF treatment on L8 cell protein synthesis. L8 myotubes
were incubated with three CNTF concentrations (0, 1, 10 ng/ml) for 12 hours. 3H-tyrosine
was added to the media during the last two hours of incubation in each treatment to a final
concentration of 1 tCi/ml. After the treatments, myofibrillar and non-myofibrillar proteins
were collected and radioactivity in each fraction was determined. Results are expressed as
a percent of control (0 ng/ml CNTF). " * " indicates this treatment differed significantly
(p<0.05) from control.
41
We also examined effects of CNTF on myofibrillar and non myofibrillar synthesis
following exposure to CNTF for 24 hours. CNTF had no significant effect (p=0.26) on
myofibrillar protein synthesis (Figure 7). However, the higher concentration of CNTF (10
ng/ml)affected a 8.5% reduction (p<0.05) in non-myofibrillar protein synthesis (Figure 7).
Effects of CNTF on Protein Degradation
In protein degradation experiments, L8 myotubes were labeled with 3H-tyrosine for
24 hours prior to application of the CNTF treatments which should label both long-lived and
short-lived proteins. CNTF treatments (0, 1, 10, 20 ng/ml) were added to DMEM which
contained 2 mM tyrosine to minimize the re-use of released 3H-tyrosine during CNTF
treatment. After CNTF treatment, three fractions of proteins were collected: media, a
myofibrillar protein-rich fraction and a non-myofibrillar protein-rich fraction. Radioactivity
in media fraction served as an index of total protein degradation, and the radioactivities in
myofibrillar and non-myofibrillar fractions specialized the changes. The total, myofibrillar or
non-myofibrillar protein degradation were expressed as a proportion of control (0 ng/ml
CNTF) values.
Following 12 hours of CNTF treatment, protein degradation was unaffected by CNTF
(p>0.05, data not shown). However, following 24 hours of treatment, there was a difference
among the groups (p=0.0075) in total protein degradation. Total protein degradation was
slightly reduced (p<0.05) by a low dose of CNTF (1 ng/ml; 7.6 %) but was accelerated
(p<0.05) by a high dose of CNTF (20 ng/ml; 12.6 %; Figure 8). Myofibrillar proteins
42
Protein Synthesis, 24h
Myofibrillar protein
Non-myofibrillar protein
10
1
CNTF, ng/ml
Figure 7. Effects of 24 hours of CNTF treatment on L8 cell protein synthesis. L8 myotubes
were incubated in different CNTF concentrations (0, 1, 10 ng/ml) for 12 hours. 3H-tyrosine
was added to the media during the last two hours of incubation in each treatment to the a
concentration of 1 11Cihnl. After the treatments, myofibrillar and non-myofibrillar proteins
were collected and radioactivity in each fraction was determined. Results are expressed as
a percent of control (0 ng/ml CNTF). "* " indicates this treatment differed significantly
(p<0.05) from control.
43
Total Protein Degradation
24h
*
CNTF, ng/ml
Figure 8. Effects of 24 hours of CNTF treatments on L8 cell total protein degradation. 3H­
tyrosine-prelabeled L8 myotubes were incubated in different CNTF concentrations (0, 1, 10,
20 ng/ml). After the treatments, the media were collected and radioactivity was determined.
Results are expressed as a percent of control (0 ng/ml CNTF). "* " indicates this treatment
differed significantly (p<0.05) from control.
44
were affected by CNTF (p<0.0001; Figure 9). At a low dose, CNTF did not affect
myofibrillar protein degradation. But, at a high dose, CNTF accelerated the degradation by
9.8 % (Figure 9). CNTF did not affect non-myofibrillar protein degradation (p = 0.739;
Figure 9). The data showed that CNTF accelerated protein degradation at a high dose and
the changes in degradation of the myofibrillar proteins were responsible.
Effect of CNTF on Total Protein Accretion
Total protein accretion was estimated by the total protein content in the control group
and treated groups.
Accretion reflects the balance of protein synthesis and protein
degradation. Results of the accretion are expressed as pg protein per plate. As shown in
Figure 10, CNTF significantly increased the protein accretion 6% (p=0.025). No differences
were detected in other treatments.
35-S-methionine Autoradiographv
An attempt to determine whether effects of CNTF on protein degradation resulted
from changes in stability of specific proteins, we assessed stabilities of individual protein in
CNTF-treated cells. Autoradiography was chosen as a method to screen protein stability.
Myotubes were labeled with 35S-methionine for 8 hours, after which decay of radioactivities
in the presence of various concentrations of CNTF (0, 1, 10 20 ng/ml) for 24 hours was
permitted. Both myofibrillar proteins and non-myofibrillar proteins were collected and
electrophoresized on SDS-PAGE gels and exposed to autoradiography film (Figures 11 and
12). Densitometry analysis revealed no significant difference between groups
45
Radioactivities remaining in myo- and non-myofibrillar proteins
24 hr of CNTF treatment
Myofibrillar protein
Non-myofibrillar protein
1
10
20
CNTF, ng/ml
Figure 9. Radioactivies remaining in myo- and non- myofibrillar proteins following 24 hours
of CNTF treatment. 3H- tyrosine - prelabeled L8 myotubes were incubated in different CNTF
concentrations (0, 1, 10, 20 ng/ml). After the treatments, the myofibrillar and non­
myofibrillar proteins were collected and radioactivity was determined. Results are
expressed as a percent of control (0 ng/ml ). "*" expressed as same as in Figure 8.
46
Total Protein Accretion
900
850
800 ­
750 ­
700
20
CNTF, ng/ml
Figure 10. Effects of 24 hours of CNTF treatment on L8 cell total protein accretion. L8
myotubes were incubated in different CNTF concentrations (0, 1, 10, 20 ng/ml) for 24 hours.
After the treatments, all the proteins were collected and total protein content was assessed.
Results are expressed as lAg protein per plate. "*" indicates the treatment differed
significantly from others (p<0.05).
47
for the visible bands (p>0.05; Figure 11 and 12). Differences in stabilities of individual
proteins were not detected (p>0.05) in muscle cells which were exposed to 12 hours of CNTF
treatment (data not shown). These data indicate that the CNTF effect on myofibrillar protein
degradation may be a general effect, not directed towards specific myofibrillar proteins.
CNTFRa Northern Blot
To evaluate whether a low expression of CNTFRa mRNA could account for the
limited effects of L8 cells to CNTF, the alteration of CNTFRcc mRNA concentration was
studied during CNTF treatment. A CNTFRa Northern blot was completed using 32P radio-
labeled DNA. Total RNA (40 p,g) extracted from CNTF-treated L8 myotube was applied
and the 32P-labeled CNTFRa fragment was used as probe. Unfortunately, this blot didn't
express a clear band (Figure 13). a-Actin probe was re-hybridized on the same membrane
as a positive control and, as expected, the blot showed clear strong bands (Figure 13). The
results indicated that the CNTFRa mRNA expression might be very limited in the L8 muscle
cell line.
RT-PCR
RT-PCR was used as a more sensitive strategy to detect CNTFRa mRNA expression.
The first strand cDNA synthesis was performed from total RNA using oligo(dT)18 as the
primer for the reverse transcription reaction. In addition to the 20 1..tg RNA from CNTF
treated L8 myotube, 5 jig of total RNA from rat muscle tissue was analyzed as positive
48
control. First PCRs were carried out by CNTFRa-specific primers which generated a PCR
product of 367 by (Figure 5). This fragment covered one intron which helped to eliminate
the contamination of genomic DNA. Figure 14 (left panel) shows that CNTFRa mRNA in
L8 cells is expressed at a low concentration when compared to skeletal muscle. A second
(nested) PCR analysis was performed with primer 1 and primer 2 which results a 229 by
fragment. This was completed in order to confirm that the first PCR product was, indeed,
generated from CNTFRa mRNA. Results identified that the 367 by 1st PCR products were
CNTFRa fragment (Figure 14, right).
This result confirms that the CNTFRa mRNA
expression is very limited in the L8 muscle cell line.
49
Figure 11. "S-methionine autoradiography of myofibrillar proteins. Myotubes pre-labeled
with "S-methionine/cysteine were incubated in control and in CNTF media for 24 hours.
Myofibrillar proteins were recovered and electrophoresed on a 7.5% - 15 % gradient SDS­
PAGE gel. The gel was dried and exposed to an autoradiography film for one day. The lanes
from the left to the right are: 1, 2, 3: CNTF 0 ng/ml (control); 4, 5, 6: 1 ng/ml; 7, 8, 9: 10
ng/ml; and 10, 11, 12: 20 ng/ml.
50
1
2
3
4
5
6
7
8
9
10
11
12
Figure 12. "S-methionine autoradiography of non-myofibrillar proteins. Myotubes pre-
labeled with "S-methionine/cysteine were incubated in control and in CNTF media for 24
hours. Non-myofibrillar proteins were recovered and electrophoresed on a 7.5% - 15 %
gradient SDS-PAGE gel. The gel was then dried and exposed to an autoradiography film for
overnight. The lanes from the left to the right are: 1, 2, 3: CNTF 0 ng/ml (control); 4, 5, 6:
1 ng/ml; 7, 8, 9: 10 ng/ml; and 10, 11, 12: 20 ng/ml.
51
4
5
7
6
?,
28S
CNTFRa
18S
11
2
3
4
5
6
7
8
9
10
28S
18S-
a -actb
Figure 13. Northern blot of CNTFRa mRNA in L8 cells. Myotubes were incubated in
control and in CNTF supplemented media for 12 hours. The RNAs were extracted and
electrophoresed on a 1.1% formaldehyde agarose gel. Upper panel: RNAs were hybridized
to CNTFRa probe. Lower panel: after stripping wash, sample RNAs were re-hybridized to
an cc-actin probe. The lanes from the left to the right are: 1, 2: CNTF 0 ng/ml (control); 3,
4: 1 ng/ml; 5, 6: 10 ng/ml; 7, 8: 20 ng/ml; and 9, 10: RNAs from rat muscle.
52
Figure 14. Results of RT-PCR analysis of CNTFRa expression. Left panel: PCR reactions
were carried out using CNTFRa -specific primers (primer 1 and primer 3) which generate a
PCR product of 367 bp. The lanes from the left to the right are 1: DNA molecular weight
marker; 2: RNA from L8 cells; 3: RNA from rat muscle; 4: negative control; 5: positive
control. Right panel: Nested PCR reactions were carried by CNTIqta -specific primers
(primer 1 and primer 2) which generate a PCR product of 229 bp. 1: DNA molecular marker;
2: PCR products from lane 2 in left panel; 3: PCR products from lane 3 in left panel; 4:
positive control; 5 :negative control. Positive controls was PCR reactions using CNTFRa
cDNA as templet. Negative control was that PCR reactions were carried without cDNA
templet.
53
DISCUSSIONS
Effects of CNTF on Muscle Protein Turnover
Opposing effects of CNTF on muscle have been reported. In denervated muscle,
CNTF exerts myotrophic effects by attenuating the skeletal muscle weight loss and
morphological changes associated with denervated atrophy (Helgren et al., 1994).
Conversely, CNTF caused cachectic wasting by speeding the catabolism of adipose tissue and
skeletal muscle (Henderson et al., 1996). Considering the complex environmental factors
which are present in in vivo studies (e.g. differences in food intake and neuronal stimuli) and
CNTF's short half-life in circulation (Dittrich et al., 1994), it is difficult to understand the
basis for CNTF's disparate effects on muscle. Myogenic cell culture provides a simpler
system to investigate CNTF action. Using an L8 cell line, our results showed that this
neuroproietic cytokine exerts different effects on protein turnover depending on its
concentration.
In this study, CNTF showed myotrophic function at low dose treatment (1 ng/ml).
The effects resulted from changes in both protein synthesis and protein degradation, and
myofibrillar proteins are responsible for the changes. In protein synthesis experiments, CNTF
(1 ng/ml) increased labeled amino acid incorporation rate into myofibrillar but not non­
myofibrillar protein synthesis following twelve hours of CNTF treatment. In protein
degradation experiments, CNTF (1 ng/ml) reduced total protein degradation. These changes
in protein synthesis and protein degradation contributed to an increase in protein accretion
at this dose. Because myofibrillar proteins are more sensitive to endocrine regulation (Bates
54
et al., 1993), it is not surprising to find that myofibrillar proteins were regulated by CNTF.
The data from the low dose CNTF treatment confirmed the myotrophic results from
denervated muscle (Helgren et al., 1994), and provides an understanding of the mechanism
of CNTF's myotrophic effects.
It is known that CNTF plays a role in promoting neuronal cell differentiation. It was
questioned whether the increase of myofibrillar protein synthesis was the result of increased
myogenic cell differentiation. To address the possibility, L8 cells were treated with CNTF
during the differentiation stage. However, there were no morphological differences between
the CNTF-treated group (1 ng/ml) and control groups (data not shown). This indicates that
the newly synthesized protein is due to the addition to the myofibrillar protein pool and not
the result of increased cell differentiation. This may be an advantage for clinical application.
An opposite catabolic effect was found at high doses of CNTF treatment (10 ng/ml).
With concentrations of z 10 ng/ml, CNTF produced protein wasting. Non- myofibrillar protein
synthesis was reduced at high concentration of CNTF and protein degradation was
significantly increased. Major losses of cell protein occurred from the myofibrillar protein
pool. The results agree with data reported by Henderson et al (1994) who suggested that a
"threshold" of active CNTF was required to cause a cachectic effect when present in
peripheral circulation at concentrations of 5-10 ng/ml in an in vivo study. This is at least 25­
fold higher than CNTF present in normal circulation (less than 0.2 ng/ml in murine sera,
Henderson et al., 1994). Using 35S-autoradiography, it was determined that CNTF effects
were general rather than directed towards specific proteins. However, it is not clear which
proteolysis mechanism was responsible for the accelerated degradation in this study. With
55
respect to the increased in protein synthesis, CNTF could participate in muscle remodeling
during the inflammatory myopathies. The results presented here help to elucidate the
physiological role of CNTF in pathologic muscle wasting.
Profound effects of CNTF have also been shown in adipose tissue. CNTF was
reported to induce hyperlipidemia by increasing both hepatic fatty acid synthesis and lipolysis.
They are regarded as a beneficial results of the acute phase response caused by infection,
inflammation and trauma (Nonogaki et al., 1996; Hardardottir et al., 1994). On the other
hand, the serum triglyceride concentration was diminished and body adipose tissue was
eliminated in CNTF-induced cachexic animals (Henderson et al., 1996). These diverse results
are similar to the effects of CNTF on muscle. A plausible explanation was that the trophic
action triggered in response to infection or injury may be involved in tissue repair before the
activation of cachexia or wasting (Nonogaki et al., 1996).
However, the regulatory
mechanism remains unknown.
CNTFRa,
It is known that CNTFRa is expressed at high levels in skeletal muscle (Davis et al.,
1993). This specific receptor is required for binding of CNTF to initiate biological responses.
Mice lacking CNTFRa display severe motor neuron deficits at birth (DeChiara et al., 1995),
but CNTF mutant mice don't exhibit notable abnormalities. This indicates that CNTFRa is
critical for CNTF function. Therefore, we determinated the receptor expression in L8 cell line
to gain insight into the initiation of CNTF's action.
56
Demonstration of CNTF receptor in L8 cells was difficult. Northern blot techniques
were first attempted; however, the CNTFRa mRNA expression in the L8 cell line was too
low to be detected by this method. A highly-sensitive technique, RT-PCR was then applied.
Twenty p.g of total RNA from L8 cells were applied in the RT-PCR reaction. Compared with
the expression in muscle (starting from 5 p.g of total RNA), CNTFRa expression was much
lower in L8 than in muscle tissue. However, the data are insufficient for quantitative
comparison. The low receptor expression in L8 cell line may be a major reason for the limited
CNTF effect in this myogenic cell line. Since little is known about the regulation of CNTF
and CNTFRa expression, further studies as needed to examine the inducibility of the receptor
in response to CNTF treatment. These studies could be conducted using quantitative RT­
PCR. These studies could clarify the mechanism by which CNTF regulates muscle protein
homeostasis by studying CNTF's myotrophic or catabolic effects in cells over-expressing
CNTFRa.
Much remains to be learned about the CNTF-receptor-signal transduction pathway.
There is no evidence so far which shows the effects of CNTF on muscle are direct results
through its JAK-STAT signal transduction pathway. CNTF was reported to increase
activated Ras in a neuroblastoma cell line (Schwarzchild et al., 1994), which functions in
regulation of proliferation, growth and differentiation. Furthermore, JAK-STAT signal
transduction pathway might "cross-talk" with other signal transduction pathways (e.g. Ras
pathway; Barr et al., 1991). Whether CNTF's effects on muscle result from signally via the
JAK-STAT pathway or via co-activating Ras-Map pathway is unclear. It will be of interest
to see the molecular regulation of CNTF in muscle cells.
57
Methodological Considerations
In this study, the effects of CNTF on muscle protein turnover were examined in
differentiated L8 myogenic cells. Unfortunately, the L8 cell line is not a stable cell line. L8
myogenic cell line was first reported in 1970 (Richler and Yaffe, 1970). The myoblasts
undergo several cycles of cell proliferation and then fuse into multinucleated myotubes when
the environment is suitable for differentiation. Several factors have been found to affect on
cell differentiation, including growth factors, myogenic factors and cooperation among cells
(Alberts, 1994). Also, the L8 cell line is known to have an unstable fiber formation manner
and it has been suggested that the culture should be recloned after several serial passages
(ATCC, 1992). It has long been known that muscle cell protein composition and protein
synthesis rate vary between stages of differentiation (Ha et al., 1979). Our observations also
showed that it was important to use consistently differentiated cells to get repeatable results.
The unstable differentiation rate may be the main factor to cause the variation among repeated
experiments.
When these studies were began, there was concern about the CNTF content in the
serum that was used in L8 cell culture. At that time, the normal CNTF plasma concentrations
were not established. L8 cells were stimulated to differentiate by reducing the sera in culture
medium (10 % fetal bovine serum in proliferation medium to 2 % horse serum in
differentiation medium). The CNTF was applied in the differentiation medium in the dose
treatment from 1 to 20 ng/ml. CNTF was found in low concentration in sera (less than 0.2
ng/ml in murine sera, Henderson et al., 1994). Hence, it is not necessary to considering the
58
limited effect of CNTF which was caused by the remaining CNTF in sera fifty-times-diluted
culture medium. There is no considerable endogenous CNTF protein in this study since the
muscle has been reported to be not the synthesis organ for CNTF (for review, see Sendtner
et al., 1994). The restricted and simple cell culture system provided an advantage to focus
on CNTF's effects on muscle protein turnover.
Certainly, there are some limitations of the method applied here. In this study, it was
demonstrated that CNTF exerts profound effects on L8 muscle cells, changing both protein
synthesis and protein degradation. Although all the effects of CNTF on muscle cell protein
turnover was small
10 %, compared the up to 50% effects in denervated muscle, Helgren
et al., 1994; and up to 25 % body weight lost in CNTF-secreting-cell implanted animal,
Henderson et al., 1994), the effects were measured within a limited time. It was wondered
if CNTF cause larger effects when the experimental duration is elongated. However, our cell
culture system is not suitable for long incubation. Another limitation in this study was about
the method which were applied that were insufficient in detecting individual protein changes
in protein synthesis and degradation. It will advance the understanding of CNTF's effects on
muscle by determining the detail change of the muscle protein turnover and the regulation in
signal transduction pathway.
In summary, it is believed that CNTF exerts mild myotrophic effects at low CNTF
concentrations but also induce muscle wasting in high concentrations. The CNTF-induced
muscle protein turnover was mediated by control of both protein synthesis and protein
degradation. The mild myotrophic function might not be a major regulator of muscle growth.
Instead, CNTF likely plays a role in body re-building in regeneration after injury, in
59
inflammation or stress causing activation of immune cytokines. Taken together with the
finding in adipose tissue studies in vivo, in which CNTF induced lipid metabolism, CNTF
might exert some trophic function during tissue repair that accompanies the acute phase
response. Our study presented here provides a more specific model in which to examine the
phenomenon in vivo.
60
CONCLUSION
CNTF was reported to exert myotrophic effects in denervated muscle but also is found
to cause muscle wasting in vivo. In this study, we confirmed these findings in the L8
myogenic cell line. At low doses of CNTF, the myofibrillar protein synthesis rate was
increased and total protein degradation was reduced resulting in an increase in total protein
accretion. High doses of CNTF accelerated myofibrillar protein degradation. These findings
suggest that the diverse effects of CNTF on muscle cells depends on CNTF concentration.
The limited effects of CNTF on protein turnover (less than 10 % in all the analysis) may result
from limited CNTF-specific receptor, CNTFRa, expression in the L8 cell line. To clarify this
assumption, further work with overexpression of the CNTFRa in a myogenic cell could be
useful. Overall, the mechanism of these effects will be important, both in understanding the
biological function of CNTF and in the clinical application of this neurotrophic factor.
61
BIBLIOGRAPHY
Adler, R., Landa, K. B., Manthorpe, M. and Varon, S. (1979). Cholinergic
neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons.
Science 204:1434-1436.
Allen, R. E. (1988). Muscle cell growth and development. In: Designing Foods Animal
Product Options In The Market Place. National Research Concil, National Acad. Press.,
Washington D. C. p 142-162.
ALS CNTF Treatment Study Group (1996). A double-blind placebo-controlled clinical
trial of subcutaneous recombinant human ciliary neurotrophic factor (rHCNTF) in
amyotrophic lateral sclerosis. Neurology 46: 1244-1249.
Arakawa, Y., Sendtner, M., and Thoenen, H. (1990). Survival effect of ciliary
neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: comparison with
other neurotrophic factors and cytokines. J. Neurosci. 10: 3507-3515.
Argiles, J. M. And Lopez-Soriano, F. J. (1996). The ubiquitin-dependent proteolytic
pathway in skeletal muscle: its role in pathological states. MS, 17:223-226.
ATCC Catalogue of cell lines & hybridomas, 7th Ed. 1992, p169
Barbin, G., Manthorpe, M. and Varon, S. (1984). Purification of the chick eye ciliary
neurotrophic factor. J. Neurochem. 43: 1468-1478.
Barr, L. F., Mabry, M., Nelkin, B. D., Tyler, G., May, W. S., and Baylin, S. B. (1991).
c-Myc gene-induced alterations in protein kinase C expression: a possible mechanism
facilitating myc-ras gene complemetation. Cancer Res. 51: 5514-19
Bates, P. C. and Millward, D. J. (1983). Myofibrillar protein turnover. Biochem. J. 214:
587-592.
Bazan, J. F. (1990). Structure design and molecular evolution of a cytokine receptor
superfamily. Proc. Natl. Acad. Sci. USA 87: 6934-6938.
Bazan, J. F. (1991). Neuropoietic cytokines in the hematopoietic fold. Neuron 7: 197-208.
Bellido, T., Stahl, N., Farruggella, T. J., Borba, V., Yancopoulos, G. D., Manolagas, S. C.
(1996). Detection of receptors for interleukin -6, interleulcin-11, leukemia inhibitory factor,
oncostatin M, and ciliary neurotrophic factor in bone marrow stromal/osteoblastic cells. J.
Clin. Invest. 97(2): 431- 437.
62
Betz, W. (1976). The formation of synapses between chick embryo skeletal muscle and
ciliary ganglia grown in vitro. J. Physiol. 254: 63-73.
Bianchi, L. M. And Cohan, C. S. (1993). Effects of the neurotrophins and CNTF on
developing statoacoustic neurons: comparison with an otocyst-derived factor. Dev. Biol.
159: 353-365.
Bloch-Gallego, E., Huchet, M., El M' Hamdi, H., Xie, F. K., Tanaka, H. and Henderson
C. E. (1991). Survival in vitro of motoneurons identification of purified by novel antibodybased methods selectively enhanced by muscle-derived factors. Development 111:221­
232.
Bonni, A., Frank, D. A., Schindler, C., and Greenberg, M. E. (1993). Charactrization of a
pathway for ciliary neurotrophic factor signaling to the neucleus. Science 262: 1575-1579.
Busch, W. A., stromer, M. H., Goll, D. E., and Suzuki, A. (1972). Ca++-specific removal
of Z-lines from rabbit skeletal muscle. J. Cell. Biol. 52: 367-381.
Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid
guanidium thiocyanate-phenol-chloroform extration. Anal. Biochem. 162: 156-159.
Ciechanover, A. (1994).The ubiquitin-proteasome proteolytic pathway. Cell 79:13-21.
Clatterbuck, R. E., Price, D. L., and Koliatsos, V. E. (1993). Ciliary neurotrophic factor
prevents retrograde neuronal death in the adult central nervous system. Proc. Natl. Acad.
Sci. USA 90: 2222-2226.
Commella, J. X., Sanz-Rodriguez, C., Aldea, M. and Esquerda, J. E. (1994). Skeletal
muscle-derived trophic factors prevent motoneurons from entering an active cell death
program in vitro. J. Neurosci. 14:2674-2686.
Conover, J. C., Ip, N. Y., Poueymirou, W. T., Bates, B., Goldfrab, M. P., DeChiara, T.
M., and Yancopoulos, G. D. (1993). Ciliary neurotrophic factor maintains the
pluripotentiality of embryonic stem cells. Development 119: 559-565.
Coux, 0., Tanaka, K., Goldberg, A. L. (1996). Structure and functions of the 20S and
26S proteasomes. Ann. Rev. Biochem. 65:801-847.
Creighton, T. E. (1993). Proteins: structure and molecular properties.p465-470 W. H.
Freeman and Company, N. Y.
Cullen, M. J., Appleyard, S. T. and Bindiff, L. (1979). Morphological aspects of muscle
breakdown and lysosomal activation. Ann NY Acad Sci 317:440-464.
63
Darnell, J. E. (1996). The JAK-STAT pathway: summary of initial studies and recent
advances. Rec. Prog. Hormone Res. 51: 391-415.
Davis, H. L. and Kierman, J. A. (1980). Neurotrophic effects of sciatic nerve extract on
denervated extensor digitorum longus muscle in the rat. Exp. Neurol. 69:124-134.
Davis, S., Aldrich, T. H., Stahl, N., Pan, L., taga, T., Kishimoto, T., Ip, N. Y., and
Yancopoulos, G. D. (1993). LIFR13 and gp130 as heterodimerizing signal transducers of
the tripartite CNTF receptor. Science 260:1805-1808.
Davis, S., Aldrich, T. H., Valenzuela, D. M., Wong, V., Furth, M. E., Squinto, S. P., and
Yancopoulos, G. D.(1991). The receptor for ciliary neurotrophic factor. Science 253: 59­
63.
Davis, S., Aldrich, T. H., Ip, N. Y., Stahi, N., Scherer, S., Farruggella, T., DiStefano, P.
S., Curtis, R., Panayotatos, N., Gascan, H., Chevalier, S., and Yancopoulos, G. D. (1993).
Released form of CNTF receptor a component as a soluble mediator of CNTF responses.
Science 259: 1736-1739.
DeChiara, T. M., Vejsada, R., Poueymirou, W. T., Acheson, A., Suri, C., Conover, J. C.,
Friedman, B., McClain, J., Pan, L., Stahl, N., Ip, N. Y., Kato, A., and Yancopoulos, G.
D. (1995). Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound
motor neuron deficits at birth. Cell 83: 313-322.
DeSerio, A., Graziani, R., Laufer, R., Ciliberto, G., and Paonessa, G. (1995). In vitro
binding of ciliary neurotrophic factor to its receptors: evidence for the formation of an IL­
6 type hexameric complex. J. Mol. Biol. 254: 795-800.
Di-Marco, A., Gloaguen, I., Graziani, R., Paonessa, G., Saggio, I., Hudson, K.R. and
Laufer, R. (1996). Identification of ciliary neurotrophic factor (CNTF) residues essential
for leukemia inhibitory factor receptor binding and generation of CNTF receptor
antagonists. Proc. Natl. Acad. Sci. USA 93: 9247-9252.
Dice, J. F. (1990). Peptide sequences that target cytosolic proteins for lysosomal
proteolysis. TIES, 15:305-309.
DiStefano, P. S., Boulton, T. G., Stark, J. L., Zhu, Y., Adryan, K. M., Ryan, T. E., and
Lindsay, R. M. (1996). Ciliary neurotrophic factor induces down-regulation of its receptor
and desensitization of signal transduction pathways in vivo: non-equivalence with
pharmacological activity. J. Biol. Chem. 271: 22839- 22846.
Dittrich, F., Thoenen, H., and Sendtner, M. (1994). Ciliary neurotrophic factor:
pharmacokinetics and acute-phase response in rat. Ann. Neurol. 35: 151-163.
64
Dubowitz, V. (1967). Cross-innervated mammalian skeletal muscle: histochemical,
physiological and biochemical observations. J. Physiol. 193:481-496.
Ebendal, T., Olson, L., Seiger, A. and Hedlund, K. (1980). Nerve growth factors in the rat
iris. Nature 286: 25-28.
Engel, A. G. and stonnington H. H. (1974). Morphological effects of denervation of
muscle. A quantilitative ultrastructural study. Ann. N. Y. Acad. Sci. 228:68-81.
Ernsberger, U., Sendtner, M., and Rohrer, H. (1989). Proliferation and differentiation of
embryonic chick sympathetic neurons: effects of ciliary neurotrophic factor. Neuron 2:
1275-1284.
Espat, N. J., Auffenberg, T., Rosenberg, J. J., Rogy, M., Martin, D., Fanf, C. H.,
Hassenlgren, P.O., Copeland, E. M., and Moldawer, L. L. (1996). Ciliary neurotrophic
factor is catabolic and shares with IL-6 the capacity to induce an acute phase response.
Am. J. Physiol. 271: R185-190.
Fagan, J. M., Waxman, L. and Goldberg A. L. (1987). Skeletal muscle and liver contain a
soluble ATP + ubiquitin-dependent proteolytic system. Biochem. J. 243:335-343.
Ferguson, J. H. (1985). Mammalian Physiology. Charles E. Merrill Publishing Company,
p132-143.
Fernandelz, H. L. and Donoso, J. A. (1987). Nerve-muscle cell trophic communication:
Introductory remarks. Chapter 1 in H. L. Fernandez and J. A. Donoso, Eds., Nervemuscle cell trophic communication. p6-13 CRC press, Boca Raton, FL.
Forger, N. G., Roberts, S. L., Wong, V., and Breedlove, S. M. (1993). Ciliary
neurotrophic factor maintains motoneurons and their target muscle in developing rats. J.
Neurosci. 13: 4720-4726.
Forger, N. G., Wong, V., and Breedlove, S. M. (1995). Ciliary neurotrophic factor arrests
muscle and motoneuron degeneration in androgen-insensitive rats. J. Neurobiol. 28: 354­
362.
Fulk, R. M., Li, J. B. and Goldberg, A. L., (1975). Effect of insulin, glucose and amino
acid on protein turnover in rat diaphragm. J. Biol. Chem. 250:290-298.
Funakoshi, H., Belluardo, N., Arenas, E., Yamamoto, Y., Casabona, A., Persson, H. and
Ibanez C. F. (1995). Muscle derived neurotrophin-4 as an activity-dependent trophic
signal for adult motor neurons. Science 268: 1495-1499.
65
Furono, K., Goodman, M. N. and Goldberg, A. L. (1990). Role of different proteolytic
systems in the degradation of muscle proteins during denervation atrophy. J. Biol. Chem.
265(15): 8550-8557.
Gearing, D. P., Thut, C. J., Vandenbos, T., Gimpel, S. D., Delaney, P. B., King, J., Price,
V., Cosman, D., and Beckmann, M. P. (1991). Leukemia inhibitory factor receptor is
structurally related to the IL-6 signal transducer, gp130. EMBO J. 10: 2839-1848.
Gogos, J.A., Thompson, R., Lowry, W., Sloane, B.F., Weintraub, H. and Horwitz, M.
(1996). Gene trapping in differentiating cell lines: regulation of the lysosomal protease
cathepsin B in skeletal myoblast growth and fusion. J. Cell. Biol. 134:837-47.
Goldberg, A. L. (1995). Function of the proteasome: The lysis at the end of the tunnel.
Science 268: 522-523.
Goll, D. E., Dayton, W. R., Singh, I. and Robson, R. M. (1991). Studies of the a­
actinin/actin interaction in the Z-disk by using calpain. J. Biol. Chem. 266:8501-8510.
Goll, D. E., Kleese, W. C. and Szpacenko, A. (1989). Skeletal muscle protease and
protein turnover. In: Animal Growth Regulation, edited by D. R. Campion, G. J.
Hausman, and R. J. Martin, Plenum publishing Corp.
Goll, D. E., Thompson, V. F., Taylor, R. G. and Christiansen, J. A. (1992). Role of the
calpain system in muscle growth. Biochem. 74:225-237.
Goodman, M. N. (1994). Interleukin-6 induces skeletal muscle protein breakdown in rats.
Proc. Soc. Exp. Biol. Med., 205:182-185.
Gooley, P. R., Johnson, B. A., Marcy, A. I., Cuca, G. C., Salowe, S. P., Hagmann, W.
K., Esser, C. K., and Springer, J. P. (1993). Secondary structure and zinc ligation of
human recombinant short-form stromelysin by multidimensional heteronuclear NMR.
Biochem 32:13098-108
Gu, Q. (1997). Biochemical and molecular characteristics of selenoprotein W. Ph. D.
Thesis, Oregon State University.
Gutman, E. (1976). Neurotrophic relations. Annu. Rev. Physiol. 38:177-216.
Guyton, A. C. (1991). Chapter 6: Contraction of skeletal muscle. Textbook of Medical
Physiology, 8th ED. W. B. Saunders Company, Philadelphia, PA.
66
Ha, D. B., Boland, R., and Martonosi, A. (1979). Synthesis of the calcium transport
ATPase of sarcoplasmic reticulum and other muscle proteins during development of
muscle cells in vivo and in vitro. Biochim. Biophy. Acta 585: 165-187.
Hagg, T., quon, D., Higaki, J., and Varon, S. (1992). Ciliary neurotrophic factor prevents
neuronal degeneration and promotes low affinity NGF receptor expression in the adult rat
CNS. Neuron 8: 145-58.
Hall, A. M., Hasselgren, P. 0., Dimlich, R. V. and Fischer, J. E. (1996). Myofibrillar
proteinase, cathepsin B, and protein breakdown rates in skeletal muscle from septic rats.
Metabolism 40: 302-6.
Hardardottir, I., Grunfeld, J. T., and Feingold, P. M. (1994). Effects of endotoxin and
cytokines on lipid metbolism. Curr. Opin. Lipidol. 5: 207-215
Heck, C. S. and Davis, H. L. (1988). Effect of denervation and nerve extract on
ultrastructure of muscle. Exp. Neurol. 100:139-153.
Heim, M. H., Kerr, I. M., Stark, G. R., and Darnell, J. E. (1995). Contribution of STAT
SH2 group to specific interferon Signaling by the Jak-STAT pathway. Science 267: 1347­
1349.
Helgren, M. E., Squinto, S. P., Davis, H. L., Heck, C. S., Zhu, Y., Yancopoulos, G. D.,
Lindsay, R. M., and DiStefano, P. S. (1994). Trophic effect of ciliary neurotrophic factor
on denervated skeletal muscle. Cell 76: 493-504.
Henderson, J. T., Seniuk, N. A., Richardson, P. M., Gauldie, J., and Roder, J. C. (1994).
Systemic administration of ciliary neurotrophic factor induces cachexia in rodents. J. Clin.
Invest. 93: 2632-2638.
Henderson, J. T., Mullen, B. J., and Roder, J. C. (1996). Physiological effects of
CNTF-induced wasting. Cytokine, 8: 784-793.
Hong, D. Y. And Forsberg, N. E. (1994). Effects of serum and insulin-like growth factor I
on protein degradation and protease gene expression in rat L8 myotubes. J. Anim. Sci. 72:
2279-2288.
Hooisma, J., slaaf, D. W., Meeter E. and Stecvens, W. F. (1975). The innervation of chick
straited muscle fibers by the chick ciliary gangle in tissue culture. Brain Res. 85: 79-85.
Horvath, C. M., and Darnell, J. E. (1997).The state of the STATs: recent developments in
the study of signal transduction to the nucleus. Curr. Opin. Cell. Biol. 9: 233-239
67
Horvath, C. M., Wen, Z., and Darnell, J. E. (1995). A STAT protein domain that
determines DNA sequence recognition suggests a novel DNA-binding domain. Genes.
Dev. 9: 984-994.
Inoue, M., Nakayama, C., Kikuchi, K., Kimura, T., Ishige, Y., Ito, A., Kanaoka, M. and
Noguchi, H.(1995). Dl cap region involved in the receptor recognition and neural cell
survival activity of human ciliary neurotrophic factor. Proc. Natl. Acad. Sci. USA 92:
8579-8583.
Ip, F. C. F., Fu, A. K. Y., Tsim, K. W. K., and Ip, N. Y. (1995). Cloning of the a
component of the chick ciliary neurotrophic factor receptor: developmental expression and
down-regulation in denervated skeletal muscle. J. Neurochem. 65: 2393-2400.
Ip, F. C., Fu, A. K., Tsim, K. W., and Ip, N. Y. (1996). Differential expression of ciliary
neurotrophic factor receptor in skeletal muscle of chick and rat after nerve injury. J.
Neurochem. 67(4): 1607- 1612.
Ip, N. Y., McClain, J., Barrezueta, N. X., Aldrich, T. H., Pan, L., Li, Y., Weigand, S.J.,
Friedman, B., Davis, S., and Yancopoulos, G. D. (1993). The a component of the CNTF
receptor is required for signaling and defines potential CNTF targets in the adult and
during development. Neuron 10: 89-102.
Ip, N. Y., and Yancopoulos, G. D. (1996). The neurotrophins and CNTF: two families
of collaorative neurotrophic factors. Annu. Rev. Neurosci. 19: 491-515
Kaupmann, K., Sendtner, M., Stockli, K.A., and Jockusch, H. (1991). The gene for ciliary
neurotrophic factor (CNTF) maps to murine chromosome 19 and its expression is not
affected in the hereditary motoneuron disease `wobbler' of the mouse. Eur. J. Neurosci. 3:
1182-1186.
Kessler, J. A., Ludlam, W. H., Freidin, M. M., Hall, D. H., Michaelson, M. D., Spray, D.
C., Dougherty, M., and Batter, D. K. (1993). Cytokine-induced programmed death of
cultured sympathetic neurons. Neuron 11: 1123-1132.
Kirsch, M. and Hofmann, H. D. (1994). Expression of ciliary neurotrophic factor receptor
mRNA and protein in the early postnantal and adult rat nervous system. Neurosci. Let.
180: 163-166.
Kirschner, R. J. And Goldberg, A.L. (1983). A high molecular weight
metalloendoprotease from the cytosol of mammalian cells. J. Biol. Chem. 254: 967-976.
Koizumi, T. (1974). Turnover rates of structural proteins of rabbit skeletal muscle. J.
Biochem. 76:431-439.
68
Kruttgen, A., Grotzinger, J., Kurapkat, G., Weis, J., Simon, R., Thier, M., Schroder, M.,
Heinrich, P., Wollmer, A., Comeau, M., Mullberg, J., Rose-John, S. (1995). Human ciliary
neurotrophic factor: a structure-function analysis. Biochem J. 309:215-20.
Kumamoto, T., Ueyama, H. Wattanabe, S., Yoshioka, K., Miike, T., Go 11, D. E., Ando,
M. and Tsuda, T. (1995). Immunohistochemical study of calpain and its endogenous
inhibitor in the skeletal muscle of muscular dystrophy. Acta Neuropathol. 89:399-403.
Kuo, W.L., Montag, A. G., Rosner, M. R. (1993). Insulin-degrading enzyme is
differentially expressed and developmentally regulated in various rat tissues.
Endocrinology, 132:604-11.
Kurek, J. B., Nouri, S. Kannourakis, G., Murphy, M., and Austin, L. (1996). Leukemia
inhibitory factor and interleukin-6 are produced by diseased and regenerating skeletal
muscle. Muscle-Nerve. 19: 1291-1301.
Kurek, J. B., Austin, L., Cheema, S.S., Barlett, P. F., and Murphy, M. (1996). Upregulation of leukemia inhibitory factor and interleukin-6 in transected sciatic nerve and
muscle following denervation. Neuromus. Disord. 6: 105-114.
Labeit, S. And Kolmerer, B. (1995). Titins: proteins in charge of muscle ultrastructure and
elasticity. Science 270:293-296.
Lam, A., Fuller, F., Miller, J., Kloss, J., Manthorpe, M., Varon, S., and Cordell, B.(1991).
Sequences and stuctural organization of the human gene encoding ciliary neurotrophic
factor. Gene 102: 271-276.
Lin, L. H., Mismer, D., Lile, J. D., Armes, L. G., Butler, E. T., Vannice, J. L. and Collins,
F. (1989). Purification, cloning, and expression of ciliary neurotrophic factor (CNTF).
Science 246:1023-1025.
Lis, J. P. (1980). Fractionation of DNA fragments by polythylene glycol induced
precipitation. Methods Enzymol. 65:347-353.
Louis, J. C., Magal, E., Burnham, P., Varon, S. (1993). Cooperative effects of ciliary
neurotrophic factor and norpinephrine on tyrosine hydroxylase expression in cultures rat
locus coeruleus neurons. Dev. Biol. 155: 1-13.
Low, R. B. and Goldberg, A. L. (1973). Non-uniform rates of turnover of myofibrillar
proteins in rat diaphragm. J. Biol. Chem. 56: 590.
69
Lowell, B.B., Ruderman, N. B. and Goodman, M. N. (1986). Evidence that lysosomes are
not involved in the degradation of myofibrillar proteins in rat skeletal muscle. Biochem. J.
234:237-240.
MacLennan, I. S. (1994). Neurogenic and myogenic regulation of skeletal muscle
formation; a critical re-evaluation. Prog. Neurobiol. (1994) 44: 119-140.
MacLennan, A. J., Vinson, E. N., Marks, L., McLaurin, D. L., Pfeifer, M., and Lee, N.
(1996). Immunohistochemical location of ciliary neurotrophic factor receptor a
expression in the rat nervous system. J. Neurosci. 16: 621-630.
MacLennan, P. A., Brown, R. A. and Rennie, M. J. (1987). A positive relationship
between protein synthetic rate and intracellular glutamine concentration in perfused rat
skeletal muscle. FEBS Lett. 215: 187-191.
Magal, E., Burnham, P., Varon, S. (1991). Effect of CNTF on low-affinity NGF receptor
expression by cultured neurons from different rat brain regions. J. Neurosci. Res. 30: 560­
566.
Maltin, C. A., Delday, M. I., Campbell, G. P. and Hesketh, J. E. (1993). Clenbuterol
mimics effects of innervation on myogenic regulation factor expression. Am. J. Physiol.
265: E176-E178.
Manthorpe, M., Skaper, S. D., Barbin, G., and Varon, S. (1982). Cholinergic
neuronotrophic factors. Concurrent activities on certain nerve growth factor-responsive
neurons. J. Neurochem. 38: 415-421.
Markelonis, G. L., and Oh, T. H. (1979). A sciatic nerve protein has a trophic effect on
development and maintenance of skeletal muscle cells in culture. Proc. Natl. Acad. Sci.
USA 76: 2470-2474.
Martin, D., Merkel, E., Tucker, K. K., McManaman, J. L., Albert, D., Relton, J., and
Russell, D. A. (1996). Cachectic effect of ciliary neurotrophic factor on innervated skeletal
muscle. Am. J. Physiol. 271: R1422-1428.
Masiakowski, P., Liu, H., Radziejewski, C., Lottspeich, F., Oberthuer, W., Wrong, V.,
Lindsay, R. M., Furth, M. E., and Panayotatos, N. (1991). Recombinant human and rat
neurotrophic factors. J. Neurochem. 57: 1003-1012.
Masu, Y., Wolf, E., Holtmann, B., Sendtner, M., Brem, G., and. (1993). Distruption of
the CNTF gene results in motor neuron degeneration. Nature 365: 27-32.
70
McComas, A. J. (1996). Skeletal muscle form and function. Human Kinetics publication.
p 4-19, 227-293, Human Kinetics, Champain, IL.
McDaniel, T., and Meltzer, S. J. (1996). Direct radioactive labeling of polymerase chain
reaction products. Methods Mol. Biol. 58: 325-327.
Medina, R., Wing, S. S. and Goldberg, A. L.(1995). Increase in levels of polyubiquitin and
proteasome mRNA in skeletal muscle during starvation and denervation atrophy.
Biochem. J. 1995 307: 631-637.
Millward, D.C. (1980). Protein degradation in muscle and liver. Comp. Biochem.
19B:153-232.
Mitsumoto, H., Ikeda, K., Klinkosz, B., Cedarbaum, J. M., Wong, V., and Lindsay, R.
M.(1994). Arrest of motor neuron disease in wobbler mice co-treated with CNTF and
BDNF. Science 265: 1107-1110.
Negro, A., Corona, G., Bigon, E., Martini, I., Grandi, C., Skaper, S. D., and Callegaro, L.
(1991). Synthesis, purification, and characterization of human ciliary neurotrophic factor
from E. Coli. J. Neurosci. Res. 29: 251-261.
Nishi, R. And Berg, D. K. (1977). Dissociated ciliary ganglion neurons in vitro: survival
and synapse formation. Proc. Natl. Acad. Sci. USA 74: 5171-5175.
Nonogaki, K., Pan, X. M., Moser, A. H., Shigenaga, J., Staprans, I., Sakamoto, N.,
Grunfeld, C., and Feingold, K. R.(1996). LIT' and CNTF, which share the gp130
transduction system, stimulate hepatic lipid metabolism in rats. Am. J. Physiol. 271: E521­
528.
Okitani, A. M., Matsuishi, M., Matsumoto, T., Kamoshida, E., Sato, M., Mstsukura, U.,
Watanabe, M., Kato,H. and Fujimaki. M. (1988). Purification and some properties of
cathepsin B from rabbit skeletal muscle. Eur. J. Biochem. 171:377.
Olsen, B. R. (1996). Morphogenesis: collagen it takes and bone it makes. Curr. Biol.
6: 645-7.
Oppenheim, R. W., Prevette, D., Yin, Q. W., Collins, F., and MacDonald, J. (1991).
Control of embryonic motoneuron survival in vivo by ciliary neurotrophic factors. Science
251: 1616-1618.
Oppenheim, R. W., Prevette, D., Haverkamp, L. J., Houenou, L., Yin, Q. W. and
MacManaman, J. (1993). Biological studies of a putative avian muscle-derived
71
neurotrophic factor that prevents naturally occurring motoneuron death in vivo. J.
Neurobiol. 24: 1065-1079.
Oppenheim, R. W. (1899). The neurotrophic theory and naturally occurring motoneuron
death. Trends Neurosci. 12:252-255.
Ou, B. (1994). The role of calpains in muscle protein degradation. Ph.D. Thesis, Oregon
State University.
Ouali, A., Bechet, D. and Dransfild E. (1993). Systematic degradation of cellular proteins:
an introduction to the workshop. Biochimie (1993), 75:837-838.
Panayotatos, N., radziejewska, E., Acheson, A., Somogyi, R., Thadani, A., Hendrickson,
W. A. and McDonald, N. Q. (1995). Localization of functional receptor epitopes on the
structure of ciliary neurotrophic factor indicates a conserved, function-related epitope
topography among helical cytokines. J. Biol. Chem. 270: 14007-14014.
Panayotatos, N., Everdeen, D., Liten, A., Somogyi, R., and Acheson, A. (1994).
Recombinant human CNTF receptor oc: production, binding stoichiometry, and
characterization of its activity as a diffusible factor. Biochemistry 33:3813-3818.
Perrone, C. E., Fenwick-Smith, D., and Vandenburgh, H. H. (1995). Collagen and stretch
modulate autocrine secretion of insulin-like growth factor-1 and insulin-like growth factor
binding proteins from differentiated skeletal muscle cells. J. Biol. Chem. 270: 2099-2106.
Peters, J. M. (1994). Proteasomes: protein degradation machines of the cell. TIBS
19:377-182.
Quinn, L. S., Haugk, K. L. and Grabstein K. H. (1995). Interleukin-15: a novel anabolic
cytokine for skeletal muscle. Endocrinology 136: 3699-72.
Rajan, P., Symes, A. J., and Fink, J. S. (1996). STAT proteins are activated by ciliary
neurotrophic factor in cells of central nervous system origin. J. Neurosci. Res. 43: 403­
411.
Rao, M. S., Tyrrel, S., Ladis, S. C., and Patterson, P. H. (1992). Effect of ciliary
neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured
sympathetic neurons. Devel. Biol. 150: 281-293.
Rayment, I. and Holden, H. M. (1994). The three-dimensional structure of a molecular
motor. TIBS, 19: 129-134.
72
Rayment, I., Holden, H.M., Whittaker, M., Yohn, C. B., Kenneth, M.L., Holmes, K. C.
and Milligan, R.A. (1993). Structure of the actin-myosin complex and its implications for
muscle contraction. Science 261:58-65.
Richardon, P. M. and Ebendal, T. (1982). Nerve growth factor activities in rat peripheral
nerve. Brain Res. 246: 57-64.
Richardson, P. M. (1994). Ciliary neurotrophic factor: a review. Pharmac. Ther. 63: 187­
198.
Rivett, A. J. (1990). Intracellulr protein degradation. Essays in biochemistry, 25:39-81.
Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D. and
Goldberg, A. L. (1994). Inhibitors of the proteasome block the degradation of peptides
presented on MI-IC class I molecules. Cell, 78:761-771.
Romanul, F. C. A., and Van Der Meulen, J. P. (1967). Slow and fast muscle after cross
innervation. Enzymatic and physiological changes. Arch. Neurol. 17: 387-402.
Saadat, S., Sendtner, M., and Rohrer, H. (1989). Ciliary neurotrophic factor induces
cholinergic differentiation of rat sympathetic neurons in culture. J. Cell. Biol. 108: 1807­
1816.
Saido, T. C., Sorimachi, H. and Suzuki, K. (1994). Calpain: new perspectives in molecular
diversity and physiological-pathological involvement. FASEB J, 8:814-822.
Samarel, A. M. (1991): In vivo measurements of protein turnover during muscle growth
and atrophy. FASEB J. 5: 2020-2028.
Schindler, C., and Darnell, J. E. (1995). Transcriptional responses to polypeptode ligands:
the JAK-STAT pathway. Ann. Rev. Biochem. 64:621-651.
Schoenheimer, R. and Rittenberg, D. (1940). The study of intermediary metabolism of
animals with the aid of isotopes. Physiol. Rev. 20: 218.
Schooltink, H., Stoyan, T., Roeb, E., Heinrich, P. C., and Rose-John, S. (1992). Ciliary
neurotrophic factor induces acute-phase protein expression in hepatocytes. FEBS Lett.
314: 280-284.
Schwarzschild, M. A., Dauer, W. T., Lewis, S. E., Hamill, L. K., Fink, J. S., and
Hyman, S. E. (1994). Leukemia inhibitory factor and ciliary neurotrophic factor increase
activatied Ras in a neuroblastoma cell line and in sympathetic neuron cultures. J.
Neurochem. 63: 1246-1254.
73
Sendtner, M., Carroll, P., Holtmann, B., Hughes, R. A. and Thoenen, H. (1994). Ciliary
neurotrophic factor. J. Neurobiol. 25: 1436-1453.
Sendtner, M., Schmalbruch, H., Stock li, K. A., Carroll, P., Kreutzberg, G. W., and
Thoenen, H. (1992). Ciliary neurotrophic factor prevent degeneration of motor neurons in
mouse mutant progressive motor neuronopathy. Nature 358: 502-504.
Seniuk-Tatton, N. A., Henderson, J. T. and Roder, J. C. (1995). Neurons experss ciliary
neurotrophic factor mRNA in the early postnatal and adult rat brain. J. Neurosci. Res.
41:663-676.
Shamberger, R. J. (1983). Biochemistry of selenium. Plenum Press, New York.
Shapiro, L., Zhang, X. X., Rupp, R. G., Wolff, S. M., and Dinarello, C.. A. (1993). Ciliary
neurotrophic factor is an endogenous pyrogen. Proc. Natl. Acad. Sci. USA 90: 8614­
8618.
Shields, D. C., Harmon, D. L., Nunez, F., and Whitehead, A. S. (1995). The evolution of
haematopoietic cytokine/receptor complexes. Cytokine 7: 679-688.
Shinohara, K., Masanori, T. and Nakano, H., Tone, S., Ito, S. And Kawashima, S. (1996).
Apoptosis induction resulting from proteasome inhibition. Biochem. J. 317:385-388.
Solomon, V. and Goldberg, A. L. (1996). Importance of the ATP-ubiquitin-proteasome
pathway in the degradation of soluble and myofibrillar protein in rabbit muscle extracts. J.
Biol. Chem. 271: 26690-26697.
Sorimachi, H., Tsukahara, T., Okada-Ban, M., Sugita, H., Ishiura, S. and Suzuki, K.
(1995). Identification of a third ubiquitous calpain species- chicken muscle expresses four
distinct calpains. Biochem. Biophys. Acta, 1261:381-393.
Sorimachi, H., Saido, T. C. and Suzuki, K. (1994). New era of calpain research,
Discovery of tissue-specific calpains. FEB Lett. 343:1-5.
Stahl, N., and Yancopoulos, G. D.(1994). The tripartite CNTF receptor complex:
activation and signaling involves components shared with other cytokines. J. Neurobiol.
25: 1454-1466.
Stahl, N., Boulton, T. G., Farruggella, T. J., Ip, N. Y., Davis, S., Witthuhn, B. A., Quelle,
F. W., Silvennoinen, 0., Barbieri, G., Pellegrini, S., Ihle, J. N., and Yancopoulos, G. D.
(1994). Association and activation of Jak-Tyk kinase by CNTF-LIF-OSM-IL-6 13 receptor
components. Science 263: 92-95.
74
Stahl, N.,Farruggella, T. J., Boulton, T. G., Zhong, Z., Darnell, J. E., and Yancopoulos,
G. D. (1995). Choice of STATs and other substrates specified by modular tyrosine-based
motifs in cytokine receptors. Science 267:1349-1353.
Stock li, K. A., Lillien, L. E., Naher-Noe, M., Breitfeld, G., Hughes, R. A., Raff, M. C.
and Thoenen, H. (1991). Regional distribution, developmental changes, and cellular
location of CNTF-mRNA and protein in the rat brain. J. Cell. Biol. 115: 447-459.
Stockli, K. A., Lottseich, F., Sendter, M., Masiakowski, P., Carroll, P., Gotz, R.,
Lindholm, D. and Thoenen, H. (1989). Molecular cloning, expression and regional
distribution of rat ciliary neurotrophic factor. Nature. 342: 21-28.
Sugden, M. C., Howard, R. M., Munday, M. R., and Holness, M. J. (1993). Mechanisms
involved in the coordinate regulation of strategic enzymes of glucose metabolism. Adv.
Enzyme. Regul. 33: 71-95.
Sugen, P. H. and Fuller, S. J. (1991). Regulation of protein turnover in skeletal and
cardiac muscle. Biochem. J. 273: 21-37.
Taga, T. (1996). gp130, a shared signal transducing receptor component for hematopoetic
and neuropoietic cytokines. J. Neurochem. 67: 1-10.
Tamura, T., Nagy, I., Lupas, A., Lottspeich, F., Cejka, Z., Schoofs, G., Tanaka, K., Mot,
R. and Baumeister (1995). The first characterization of a eubacterial proteasome: the 20S
complex of Rhodococcus. Curr. Biol. 1995, 5:766-774.
Temparis, S., Asensi, M., Taillandier, D., Aurousseau, E., Larbaud, B., Obled, A., Bechet,
D., Ferrara, M., Estrela, J.M. and Attaix, D. (1994). Increase ATP-ubiquitin-dependent
proteolysis in skeletal muscles of tumor bearing rats. Cancer Res.54:5568-5573.
Toylor, R. G., Tassy, G., Briand, M., Robert, N., Briand, Y. and Ouali, A. (1995).
Proteolytic activity of proteasome on myofibrillar structure. Mole. Biol. Rep. 21:71-73.
Trinick, T. (1994). Titin and nebulin: protein rules in muscle? TIES, 19:405-408
Tsujinaka, T., Fujita, J., Ebisui, C., Yano, M., Kominami, E., Suzuki, K., Tanaka, K.,
Katsume, A., Ohsugi, Y., Shiozaki, H., and Monden, M. (1996). Interleukin 6 receptor
antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6
transgenic mice. J. Clin. Invest. 97: 244-249.
Valenzuela, D.M., Stitt, T. N., DiStefano, P.S., Rojas, E., Mattsson, K., Compton, D.L.,
Nunez, L., Park, J.S., Stark, J.L., Gies, D. R., Thomas, S., Le Beau, M. M., Fernald, A.
A., Burden, S. L., Glass, D. J. and Yancopoulos, G. D. (1995). Receptor tyrosine kinase
75
specific for the skeletal muscle lineage: expression in embryonic muscle, at the
neuromuscular junction, and after injury. Neuron 15:573-584.
Valenzuela, D. M., Rojas, E., Le Beau, M. M., Espinosa, R., brannan, C. I., McClain, J.,
Masiakowski, P., Ip, N. Y., Copeland, N. G., Jenkins, N. A., and Yancopoulos, G. D.
(1995). Genomic organization and chomosomal location of the human and mouse genes
encoding the a receptor component for ciliary neurotrophic factor. Genomic 25: 157-163.
Wang, K. K. and Yuen, P. W. (1994). Calpain inhibition: an overview of its therapeutic
potential. Trends Pharmacol. Sci., 15:412-419.
Wang, Y. and Fuller, G. M. (1995). Interleukin-6 and ciliary neurotrophic factor trigger
Janus kinase activation and early gene response in rat hepatocytes. Gene 162: 285-289.
Wessels, N. K., Nuttall, R. P., Wrenn, J. T. and Johnson, S. (1976). Differential labeling
of the cell surface of single ciliary ganglion neurons in vitro. Proc. Natl. Acad. Sci. USA
73: 4100-4104.
Whipple, G. and Koohmaraie, M. (1991). Degradation of myofibrillar proteins by
extractable lysosomal enzymes and m-calpain, and the enzyme of zinc chloride. J. Anim.
Sci. 69:4449-4460.
Williams, L.R., Mathorpe, M., Barbin, G., Nieto-Sampedro, M., Cotman, C. W. and
Varon, S. (1984). High ciliary neurotrophic specific activity in rat peripheral nerve. Int. J.
Dev. Neurosci. 2: 177-180.
Williams, D. B., and Windebank, A. J. (1991). Motor neuron disease (amyotrophic lateral
sclerosis). Mayo. Clin. Proc. 66:54-82.
Wing, S. S., Haas, A.L. and Goldberg, A. L. (1995). Increase in ubiquitin-protein
conjugates concomitant with the increase in proteolysis in rat skeletal muscle during
starvation and atrophy denervation. Biochem. J. 307:639-645.
Wu, G. and Thompson, J. R. (1990). The effect of glutamine on protein turnover in chick
skeletal muscle in vitro. Biochem. J. 265: 593-598.
Xu, X., Sun, Y. L., and Hoey, T. (1996). Cooperative binding and sequence-selective
recognition conferred by the STAT amino-terminal domain. Science 273: 794-797.
Yokoji, H., Ariyama, T., Takahashi, R., Inazawa, J., Misawa, H. and Deguchi, T. (1995).
cDNA cloning and chromosomal location of the human ciliary neurotrophic factor gene.
Neurosci. Let. 185: 175-178.
76
Zierath, J.R. (1995). In vitro studies of human skeletal muscle: hormonal and metabolic
regulation of glucose transport. Acta Physiol. Scand. Suppl. 626:11-96.