U.S. patent application number 09/901511 was filed with the patent office on 2001-12-06 for myostatin gene promoter and inhibition of activation thereof.
Invention is credited to Wang, Jiahong, Wu-Wong, Jinshyun R..
Application Number | 20010049435 09/901511 |
Document ID | / |
Family ID | 23286552 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010049435 |
Kind Code |
A1 |
Wu-Wong, Jinshyun R. ; et
al. |
December 6, 2001 |
Myostatin gene promoter and inhibition of activation thereof
Abstract
The subject invention relates to a promoter which induces
expression of the myostatin gene as well as to methods for
identifying compositions useful for the inhibition of the promoter,
and also methods and compositions useful for preventing the
synthesis, secretion and function of myostatin. In particular,
inhibitors that prevent the synthesis, secretion and function of
myostatin may be used to prevent the loss of muscle mass in humans
and animals.
Inventors: |
Wu-Wong, Jinshyun R.; (Lake
Bluff, IL) ; Wang, Jiahong; (Northbrook, IL) |
Correspondence
Address: |
Steven F. Weinstock
Abbott Laboratories
Department 377 / AP6D-2
100 Abbott Park Road
Abbott Park
IL
60064-6050
US
|
Family ID: |
23286552 |
Appl. No.: |
09/901511 |
Filed: |
July 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09901511 |
Jul 9, 2001 |
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09329685 |
Jun 10, 1999 |
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6284882 |
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Current U.S.
Class: |
536/24.1 ;
435/325; 435/69.1; 435/7.93 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; C07K 14/475 20130101; A61P 21/00 20180101;
A61P 31/18 20180101; A61P 25/00 20180101 |
Class at
Publication: |
536/24.1 ;
514/44; 435/7.93; 435/69.1; 435/325 |
International
Class: |
A61K 048/00; G01N
033/53; C07H 021/04; C12N 005/06; C12P 021/02 |
Claims
1. An isolated nucleic acid sequence represented by FIG. 2 (SEQ ID
NO:1).
2. A vector comprising said nucleic acid sequence of claim 1 and a
nucleic acid sequence encoding a reporter molecule, said nucleic
acid sequence encoding said reporter molecule being operably linked
to said nucleic acid sequence of claim 1.
3. The vector of claim 2, wherein said reporter molecule is
selected from the group consisting of luciferase,
.beta.-galactosidase and Chloramphenicol Acetyltransferase
(CAT).
4. The vector of claim 3 wherein said reporter molecule is
luciferase.
5. A host cell comprising said vector of claim 3.
6. A purified antibody produced in response to immunization with
myostatin.
7. A composition comprising said purified antibody of claim 6.
8. A method of identifying a composition which inhibits activation
of the myostatin promoter comprising the steps of: a) constructing
a vector comprising a nucleic acid sequence represented by FIG. 2
(SEQ ID NO:1) and a nucleic acid sequence encoding a reporter
molecule, said nucleic acid sequence encoding said reporter
molecule being operably linked to said nucleic acid sequence
encoding said sequence represented by FIG. 2; b) introducing said
vector into a host cell for a time and under conditions suitable
for expression of myostatin; c) exposing said host cell to a
composition which may inhibit activation of the myostatin promoter
and a substrate specific for said reporter molecule; and d)
measuring the signal generated by reaction of said reporter
molecule and said substrate in comparison to that produced by a
control host cell, a smaller signal by said host cell of (c)
indicating that said composition will inhibit activation of said
myostatin promoter.
9. A method of identifying a composition which inhibits expression
of myostatin comprising the steps of: a) adding an antibody
selected from the group consisting of a monoclonal or a polyclonal
antibody produced against myostatin to a solid phase; b) adding
known concentrations of myostatin or a cell sample comprising
myostatin exposed to said test composition, to said solid phase, in
order to form a first complex between said antibody and said known
concentrations of myostatin or myostatin in said cell sample; c)
adding a second antibody to said first complex, selected from the
group consisting of a monoclonal antibody or a polyclonal antibody
produced against myostatin for a time and under conditions
sufficient for formation of a second complex between said first
complex and said second antibody; d) contacting said second complex
with an indicator reagent which comprises a signal generating
compound attached to an antibody against said antibody of said
second complex, for a time and under conditions sufficient for
formation of a third complex; and e) detecting the presence of a
measurable signal, absence of said signal indicating said
composition inhibits expression of myostatin and presence of said
signal indicating said composition does not inhibit expression of
myostatin.
10. A method of identifying a composition which inhibits expression
of myostatin comprising the steps of: a) coating a fixed amount of
myostatin on a solid phase; b) adding known concentrations of
myostatin or a cell sample comprising myostatin, exposed to said
composition; c) contacting an antibody selected from the group
consisting of a monoclonal antibody or a polyclonal antibody
produced against myostatin to said myostatin in (a) and (b) for a
time and under conditions sufficient to form a first complex,
wherein myostatin of (a) competes with myostatin of (b) for said
monoclonal or polyclonal antibody; d) contacting said first complex
with an indicator reagent which comprises a signal generating
compound attached to an antibody against said antibody of said
first complex, for a time and under conditions sufficient to form a
second complex; and e) detecting a measurable signal, a higher
signal as compared to a control, indicating said composition
inhibits myostatin expression.
11. A method of identifying a composition, in a mixture of
compositions, which prevents myostatin from binding to a myostatin
receptor comprising the steps of: a) mixing purified myostatin with
said mixture of compositions; b) passing the resulting mixture of
step (a) through a filter having pores of a size such that a
composition which is complexed to myostatin does not pass through
said filter; and c) determining the structure of a complexed
composition, thereby identifying a composition which prevents
myostatin from binding to a myostatin receptor.
12. A method of identifying a composition which prevents myostatin
from binding to a myostatin receptor comprising the steps of: a)
radiolabeling recombinant myostatin; b) incubating said
radiolabeled recombinant myostatin with cells or membranes
comprising a myostatin receptor; c) contacting the incubated
mixture of step (b) with a composition of interest; d) separating
radiolabeled myostatin bound to cells or membranes from unbound
myostatin; and e) determining the amount of radioactivity in bound
myostatin, compared to a control, a lower level of radioactivity in
bound myostatin compared to said control indicating a composition
which inhibits myostatin from binding to a receptor.
13. A method of preventing muscle wasting in a mammal comprising
administering to said mammal a composition comprising an active
ingredient which causes an effect selected from the group
consisting of preventing activation of the myostatin promoter,
preventing synthesis of myostatin, and preventing myostatin from
binding to its target receptor, in a therapeutically effective
amount, such that muscle wasting is prevented.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Technical Field
[0002] The subject invention relates to a promoter which regulates
expression of the myostatin gene as well as methods of inhibiting
this promoter and compositions used for such inhibition. In
particular, inhibitors of the promoter prevent the expression of
the myostatin gene and thus prevent muscle wasting.
[0003] 2. Background Information
[0004] Myostatin, or growth/differentiation factor 8 (GDF-8),
belongs to the transforming growth factor-.beta. (TGF-.beta.)
superfamily (McPherron et al., Nature 387:83-90 (1997)). The human
myostatin gene has been cloned (Nestor et al. Proc. Natl. Acad.
Sci. 95:14938-43 (1998)), and it has been reported that myostatin
immunoreactivity is detectable in human skeletal muscle in both
type 1 and type 2 fibers. With respect to function, myostatin may
play a role in negatively regulating the growth and development of
skeletal muscle (Nestor et al., supra).
[0005] The first evidence that myostatin may play a key role in
negatively regulating muscle development came from a study with
myostatin knock-out mice (McPherron et al., Nature 387:83-90
(1997)). In the myostatin null mice, the animals were rather normal
except that they were significantly larger than wild-type mice and
had a large and widespread increase in skeletal muscle mass.
Furthermore, it was also determined that two breeds of cattle,
characterized by increased muscle mass, have mutations in the
myostatin coding sequence (McPherron et al., Proc. Natl. Acad. Sci.
94:12457-61 (1997)). Additionally, it should be noted that the
serum and intramuscular concentrations of immunoreactive myostatin
are increased in HIV-infected men with muscle wasting compared with
healthy men, and correlate inversely with the fat-free mass index.
These data support the hypothesis that myostatin is a negative
regulator of skeletal muscle growth in adult men and contributes to
muscle wasting in HIV-infected men (Nestor et al., supra).
[0006] In view of the above findings, a need exists for a manner of
regulating myostatin expression, particularly in individuals who
experience muscle wasting as a result of a condition or disease
state such as, for example, aging, Autoimmune Deficiency Syndrome
(AIDS), Multiple Sclerosis, and cancer. The present invention
provides methods and compositions which may be utilized to help
individuals with such muscle wasting conditions and provides
further insight into the regulation of myostatin gene
expression.
[0007] All U.S. patents and publications referred to herein are
hereby incorporated in their entirety by reference.
SUMMARY OF THE INVENTION
[0008] The present invention encompasses an isolated nucleic acid
sequence represented by FIG. 2 (SEQ ID NO:1).
[0009] Additionally, the present invention encompasses a vector
comprising the above-described nucleic acid sequence and a nucleic
acid sequence encoding a reporter molecule. The nucleic acid
sequence encoding the reporter molecule is operably linked to the
nucleic acid sequence represented by FIG. 2. The reporter molecule
may be selected from the group consisting of, for example,
luciferase, .beta.-galactosidase and Chloramphenicol
Acetyltransferase (CAT). Preferably, the reporter molecule is
luciferase. The present invention also includes a host cell
comprising the above-described vector.
[0010] Additionally, the present invention includes a purified
antibody produced in response to immunization with myostatin as
well as a composition comprising this purified antibody.
[0011] Furthermore, the present invention also includes a method of
identifying a composition which inhibits activation of the
myostatin promoter. This method comprises the steps of: a)
constructing a vector comprising a nucleic acid sequence
represented by FIG. 2 (SEQ ID NO:1) and a nucleic acid sequence
encoding a reporter molecule, the nucleic acid sequence encoding
the reporter molecule being operably linked to the nucleic acid
sequence encoding the sequence represented by FIG. 2; b)
introducing the vector into a host cell for a time and under
conditions suitable for expression of myostatin; c) exposing the
host cell to a composition which may inhibit activation of the
myostatin promoter and a substrate specific for the reporter
molecule; and d) measuring the signal generated by reaction of the
reporter molecule and the substrate in comparison to that produced
by a control host cell, a smaller signal by the host cell of (c)
indicating that the composition will inhibit activation of the
myostatin promoter.]
[0012] Also, the present invention includes a method of identifying
a composition which inhibits expression of myostatin comprising the
steps of: a) adding an antibody selected from the group consisting
of a monoclonal or a polyclonal antibody produced against myostatin
to a solid phase; b) adding known concentrations of myostatin or a
cell sample comprising myostatin exposed to the test composition,
to the solid phase, in order to form a first complex between the
antibody and the known concentrations of myostatin or myostatin in
said cell sample; c) adding a second antibody to the first complex,
selected from the group consisting of a monoclonal antibody or a
polyclonal antibody produced against myostatin for a time and under
conditions sufficient for formation of a second complex between the
first complex and the second antibody; d) contacting the second
complex with an indicator reagent which comprises a signal
generating compound attached to an antibody against said antibody
of said second complex, for a time and under conditions sufficient
for formation of a third complex; e) detecting the presence of a
measurable signal, absence of the signal indicating that the
composition inhibits expression of myostatin and presence of the
signal indicating that the composition does not inhibit expression
of myostatin.
[0013] Moreover, the present invention also includes a method of
identifying a composition which inhibits expression of myostatin
comprising the steps of: a) coating a fixed amount of myostatin on
a solid phase; b) adding known concentrations of myostatin or a
cell sample comprising myostatin exposed to the composition; c)
contacting an antibody selected from the group consisting of a
monoclonal antibody or a polyclonal antibody produced against
myostatin to the myostatin in (a) and (b) for a time and under
conditions sufficient to form a first complex, wherein myostatin of
(a) competes with the myostatin of (b), in a competition for the
antibody; d) contacting the complex with an indicator reagent which
comprises a signal generating compound attached to an antibody
against the antibody of the first complex, for a time and under
conditions sufficient to form a second complex; and e) detecting a
measurable signal, a higher signal as compared to a control,
indicating the composition inhibits myostatin expression.
[0014] Additionally, the present invention includes a method of
identifying a composition, in a mixture of compositions, which
prevents myostatin from binding to a myostatin receptor comprising
the steps of: a) mixing purified myostatin with the mixture of
compositions; b) passing the resulting mixture of step (a) through
a filter having pores of a size such that a composition which is
complexed to myostatin does not pass through the filter; and c)
determining the structure of a complexed composition, thereby
identifying a composition which prevents myostatin from binding to
a myostatin receptor.
[0015] Also, the present invention encompasses a method of
identifying a composition which prevents myostatin from binding to
a myostatin receptor comprising the steps of: a) radiolabeling
recombinant myostatin; b)incubating the radiolabeled myostatin with
cells or membranes comprising a myostatin receptor; c) contacting
the incubated mixture of step (b) with a composition of interest;
d) separating radiolabeled myostatin bound to cells or membranes
from unbound myostatin; and e) determining the amount of
radioactivity in bound myostatin, compared to a control, a lower
level of radioactivity in bound myostatin compared to said control
indicating a composition which inhibits myostatin from binding to a
receptor.
[0016] Furthermore, the present invention includes a method of
preventing muscle wasting in a mammal comprising administering to
the mammal a composition comprising an active ingredient which
causes an effect selected from the group consisting of preventing
activation of the myostatin promoter, preventing synthesis of
myostatin, and preventing myostatin from binding to its target
receptor, in a therapeutically effective amount, such that muscle
wasting is prevented.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 represents a conceptual illustration of the human
myostatin gene. The human myostatin gene contains three exons and
two introns, which encode 1.1 kb of myostatin cDNA. When a human
P1-derived Artificial Chromosome (PAC) library was screened using a
740 bp probe encoding human myostatin exon 1 and 2, one positive
clone with a 120 kb insert was identified. After digesting the
clone with EcoRI, two human genomic subclones were isolated. One of
the subclones, Clone C11, contained 0.37 kb of exon 1, 1.53 kb of
intron 1, and a 3.4 kb sequence containing the human myostatin
promoter region.
[0018] FIG. 2 represents the nucleic acid sequence of the human
myostatin promoter region. The putative transcription factor
binding regions are identified and underlined.
[0019] FIG. 3 represents the detection of myostatin mRNA by RT-PCR
of RNA samples from human skeletal muscle, rhabdomyosarcoma cells,
and prostate smooth muscle cells using human myostatin-specific
primers, and for G3PDH using G3PDH-specific primers. Amplified
myostatin gene (1.1 kb) was observed in both human skeletal muscle
cells (lane 1) and rhabdomyosarcoma cells (lane 2), but not in
prostate smooth muscle cells (lane 3). Amplified G3PDH gene (0.9
kb) was observed in skeletal muscle (lane 4), rhabdomyosarcoma
cells (lane 5), and prostate smooth muscle cells (lane 6).
[0020] FIG. 4 represents the luciferase reporter constructs. The
3.4 kb human myostatin promoter region was cloned into the XhoI and
HindIII sites of a luciferase reporter pGL3-enhancer vector
(Promega, Madison, Wis.), which contained a luciferase reporter
gene and a SV40 enhancer element for increasing the transcription
level.
[0021] FIG. 5 represents results from a luciferase assay for the
human myostatin promoter region in human skeletal muscle cells and
rhabdomyosarcoma cells.
DETAILED DESCRIPTION OF THE INVENTION
[0022] As noted above, the subject invention relates to the
identification and isolation of a promoter which is involved in
activating or regulating expression of the myostatin gene.
Myostatin negatively regulates the growth and development of
skeletal muscle.
[0023] In particular, the subject invention relates to methods
which may be used to identify compounds that inhibit the myostatin
promoter activities. The myostatin promoter region or nucleic acid
sequence which regulates the expression of myostatin is linked to
the luciferase reporter gene. Thus, if one is able to identify
compounds that inhibit the activity of the myostatin promoter
region to prevent expression of luciferase, one may then prevent
the promoter region from functioning, thereby preventing expression
of myostatin.
[0024] The identification of compounds which inhibit the myostatin
promoter activity and luciferase production may be carried out by
the use of drug screening assays. Initially, a vector is created
comprising an isolated DNA sequence encoding the promoter region of
myostatin, which is linked to the luciferase reporter gene. The
vector may be, for example, a plasmid, a bacteriophage or a cosmid.
The vector is then introduced into host cells under time and
conditions suitable for activation of the myostatin promoter. The
host cells may be prokaryotic or eukaryotic cells. Preferably,
eukaryotic cells are utilized, for example, cell lines with muscle
lineage. Examples include human skeletal muscle cells, human
rhabdomyosarcoma cells, and rat L6 or L8 cells. The host cells are
then exposed to the test composition thought to block activation of
the myostatin promoter and luciferase gene expression. The cells
are also exposed to a substrate for luciferase. One then measures
the quantity of signals or light emitted from the
luciferase-substrate reaction. If the amount of signals produced by
the host cells, exposed to the composition in question, is lower
than that produced by control cells (i.e., cells which have not
been exposed to the composition), then the composition has
inhibited the activity of the myostatin promoter, and will be
useful in inhibiting the expression of the myostatin gene. If the
amount of signals produced by the treated cells is equal to that
produced by the control cells, the composition has not inhibited
the activity of the myostatin promoter and will not prevent
myostatin gene expression.
[0025] Once compositions have been identified which inhibit the
activity of the myostatin promoter, such compositions may be
administered to patients having any type of condition involving
muscle wasting, for example, Acquired Immunodeficiency Syndrome
(AIDS), cancer, Multiple Sclerosis and aging. The pharmaceutical
composition may comprise a therapeutically effective amount of the
inhibitor and an appropriate physiologically acceptable carrier
(e.g., water, buffered water or saline). The dosage, form (e.g.,
suspension, tablet, capsule, etc.), and route of administration of
the pharmaceutical composition (e.g., oral, topical, intravenous,
subcutaneous, etc.) may be readily determined by a medical
practitioner and may depend upon such factors as, for example, the
patient's age, weight, immune status, and overall health.
[0026] Additionally, the present invention also encompasses
compositions comprising antibodies derived using purified myostatin
protein or a portion thereof which may be administered with, for
example, an appropriate carrier (e.g., water, buffered water or
saline). Subsequent to administration of the antibodies, they may
bind to expressed myostatin in the body in order to form a complex,
thereby preventing the expressed myostatin from negatively
regulating muscle development. The antibodies themselves, as well
as portions thereof, are also encompassed within the scope of the
present invention, as well as assays which comprise such antibodies
or portions thereof.
[0027] It should be noted that the above pharmaceutical
compositions and antibodies may be utilized for veterinary
applications (e.g., for preventing muscle wasting in aging or
diseased animals) or for agricultural applications (e.g., for
increasing the meat production in livestock, since animals without
myostatin exhibit a much larger muscle mass). For example, the
therapeutic composition which inhibits the activity of the
myostatin promoter may be administered to mammals such as, for
example, horses, cows, sheep, goats, cats, dogs and pigs.
[0028] The present invention also covers two methods, using
purified myostatin and/or myostatin antibodies, which identify
compositions that inhibit the synthesis and the secretion of the
myostatin protein. In the sandwich method, a mammalian monoclonal
and/or polyclonal antibody (e.g., rabbit or mouse) against the
mature form of myostatin is coated on a solid surface (e.g., the
Immulon-4 plate (Dynatech Laboratories INC., Chantilly, Va.)). The
surface will be blotted by a known blotting agent, for example,
Bovine Serum Albumin (BSA), and washed. Samples (e.g. supernatants
from human skeletal muscle cells treated with or without test
agents) or known concentrations of purified mature form of
myostatin are added to the surface (e.g., plate). After myostatin
binds to the antibody or antibodies, the surface will be washed,
and then incubated with a mammalian monoclonal and/or polyclonal
antibody (e.g., goat, rabbit or mouse) against myostatin. The
binding of the second anti-myostatin antibody will be detected by
use of an indicator reagent which comprises an antibody conjugated
with a signal generating compound, for example, an enzyme. A
substrate for the enzyme is also added if an enzyme is utilized.
For example, horseradish peroxidase (HRP) and its substrate
O-Phenylenediamine hydrochloride (OPD) may be utilized. In
particular, the enzyme-substrate reaction generates a detectable
signal or change, for example, color, which may be read, for
example, in a Microplate Reader. Examples of signal generating
compounds, other than an enzyme which may be utilized include, for
example, a luminescent compound, a radioactive element, a visual
label and a chemiluminescent compound. Known concentrations of
purified myostatin are used to generate a standard curve. The
concentration of myostatin in the unknown samples (e.g.
supernatants from human skeletal muscle cells treated with or
without test agents) can be determined using the standard curve.
The test agents which decrease the myostatin concentration in
supernatants are potentially useful for inhibition of myostatin
synthesis/secretion.
[0029] In the competitive method, a fixed amount of human myostatin
is coated on a solid surface, for example, the Immulon-4 plate. The
plate will be blotted by, for example, BSA or another known
blotting agent, and washed. Samples (e.g., supernatants from human
skeletal muscle cells treated with or without test agents) or known
concentrations of purified mature form of myostatin are added to
the plate along with a mammalian monoclonal and/or polyclonal
antibody (e.g., goat, rabbit or mouse) against myostatin. The plate
will be washed, and then incubated with an indicator reagent
comprising an antibody conjugated with a signal generating
compound, for example, an enzyme (or the entities described above).
If an enzyme is used, a substrate for the enzyme is also provided.
The enzyme may be, for example, horseradish peroxidase (HRP) The
substrate may therefore be O-Phenylenediamine hydrochloride (OPD)).
Again, the enzyme-substrate reaction generates a detectable change
or signal, for example, color, which can be read in, for example, a
Microplate Reader. Known concentrations of purified myostatin may
be used to generate a standard curve. The concentration of
myostatin in the unknown samples (e.g. supernatants from human
skeletal muscle cells treated with or without test agents) can be
determined using the standard curve. The test agents which decrease
the myostatin concentration in supernatants are potentially useful
for inhibition of myostatin synthesis/secretion. Known
concentrations of myostatin, or myostatin in the sample, compete
with myostatin protein coated on the plate in binding to myostatin
antibodies. When more myostatin is present in the sample, less
signal is generated. If a test agent is able to block myostatin
synthesis/secretion, the amount of myostatin in that particular
sample will be less than in the control, and the signal in that
sample will be more than in the control.
[0030] Additionally, the present invention covers an
Affinity-Selection method, using purified myostatin in a filtration
assay, to identify compositions that bind to myostatin to prevent
myostatin from binding to its receptors, thus preventing myostatin
from functioning. Briefly, purified myostatin is mixed with several
test compounds. The mixture is passed through a filter which only
allows certain molecular weight molecules to pass through.
Compositions that bind to myostatin will be retained by the filter.
The unbound compounds are not retained and can be separated from
the bound compositions. The structures of the compositions which
bind to myostatin are determined, for example, by Mass
Spectrometry.
[0031] Furthermore, the present invention also encompasses a
receptor binding method using radiolabeled myostatin to bind to
cells or membranes prepared from tissues or cells containing
myostatin receptors. In this manner, one may identify compositions
that block myostatin from binding to its receptors, thus preventing
myostatin from functioning. In particular, the purified recombinant
myostatin protein from bacteria, insect or mammalian cells is
radiolabeled ([.sup.125I], [.sup.3H], [.sup.14C], etc.). The
radiolabeled myostatin is then incubated with cells or membranes
prepared from tissues or cells which contain myostatin receptors in
the presence or absence of the test composition. Radiolabeled cells
and membranes are then separated from non-radiolabeled cells and
membranes by separation methods such as, for example, filtration
and centrifugation. The amount of myostatin binding to cells or
membranes is determined by counting radioactivity. A decrease in
radioactivity in the presence of a test composition indicates that
the composition inhibits myostatin binding, and thus is useful in
inhibiting myostatin function.
[0032] The present invention may be illustrated by the use of the
following non-limiting examples:
EXAMPLE I
Identification of the Nucleotide Sequence Encoding the Human
Myostatin Promoter and Potential Transcription Factors Binding
Regions
[0033] 1. Cloning of human myostatin cDNA. Human myostatin cDNA was
amplified from human skeletal muscle 5'-plus cDNA library
(Clontech, Palo Alto, Calif.) by PCR using specific primers, 5'-
ATG CAA AAA CTG CAA CTC TGT GTT T -3' and 5'- TCA TGA GCA CCC ACA
GCG GTC -3'. PCR products were cloned into eukaryotic TA cloning
vector pCR3.1 (Invitrogen, Carlsbad, Calif.). Insertion was
confirmed by DNA sequencing.
[0034] 2. Cloning of the human myostatin promoter region. The EcoRI
and HindIII fragment (740 bp) of human myostatin cDNA (FIG. 1),
which covers exon 1 and 2 of human myostatin gene, was sent to
GenomeSystems Inc. (St. Louis, Mo.) as a probe to screen the human
P1-derived Artificial Chromosome (PAC) library. One positive clone
with 120 kb insert was identified and confirmed by genomic Southern
blot using the same probe. The 120 kb insert was digested with
EcoRI restriction enzyme, and subcloned into plasmid pzero
(Invitrogen, Carlbad, Calif.). Two positive subclones with 5-7 kb
insert were identified (FIG. 1). Sequencing results indicate that
clone 10 contains exon 2 and part of intron 1 and 2. Clone 11 (5.3
kb) contains exon 1, part of intron 1, and a 3.4 kb 5' untranslated
region--the putative myostatin promoter region. Program
MatInspector V2.2 (Gesellschaft fur Biotechnologische Forschung
mbH, Braunschweig, Germany) was searched to identify potential
transcription factor binding regions (FIG. 2). MatInspector is a
software that allows fast scanning of sequence data for consensus
motifs. MatInspector uses the core similarity, the matrix
similarity and the Ci vector created by MatInd to calculate
similarity index. The potential transcription factor binding sites
were selected when both core similarity and matrix similarity reach
0.95. The details of the program are illustrated in Quandt et al.,
Nucleic Acids Research 23:4878-4884, 1995.
[0035] 3.Identification of Human skeletal muscle cells or other
cell lines of muscle lineage which express myostatin. Total RNA
from human skeletal muscle cells and rhabdomyosarcoma cells was
isolated using Trizol reagent (Life Technologies, Gaithersburg,
Md.) and used as templates for reverse transcription. The myostatin
gene was amplified by PCR using human myostatin specific primers,
5'- ATG CAA AAA CTG CAA CTC TGT GTT T -3' and 5'- TCA TGA GCA CCC
ACA GCG GTC -3'. Total RNA from human prostate smooth muscle cells
were also tested. Primers for endogenous G3PDH gene was used to
test the integrity of total RNA. The PCR products were analyzed on
a 1% agarose gel (FIG. 3). The 1.1 kb PCR products, 1.1 kb being
the correct size for myostatin, indicated that both human skeletal
muscle cells and rhabdomyosarcoma cells express myostatin.
EXAMPLE II
Luciferase Assay
[0036] The luciferase reporter gene assay was designed for
quantitative analysis of mammalian gene expression. The coding
region for firefly (Photinus pyralis) luciferase was linked to the
3.4 kb 5' untranslated region (5-UTR) of the human myostatin gene.
The construct was transiently transfected into human skeletal
muscle cells or human rhabdomyosarcoma cells, and after 48 hours,
luciferase activity was measured. To assay for the luciferase
activity, luciferin and Mg.sup.2+-ATP were added to cellular
extracts, and the production of light was monitored. The luciferase
activity is increased if the 3.4 kb 5'-UTR region has promoter
activity. Therefore, the luciferase activity may be used as an
indicator (reporter) of the function of the upstream promoter
region.
[0037] The 3.4 kb human myostatin 5' untranslated region was cloned
into XhoI and HindIII sites of pGL3-enhancer vector (Promega,
Madison, Wis.), linked to the luciferase reporter gene (FIG. 4).
The construct was transiently transfected into human skeletal
muscle cells or human rhabdomyosarcoma cells using Superfect
reagents (Qiagen, Inc., Santa Clarita, Calif.). Another reporter
gene such as .beta.-galactosidase gene or Renilla luciferase gene
was co-transfected as control for transfection efficiency. Cells
were lysed in lysis buffer after 48 hours, and cellular extracts
were assayed for luciferase and .beta.-gal activity using detection
kit [e.g. LucLite (Packard, Meriden, Conn.), luminescent
.beta.-galactosidase detection Kit II (Clontech, Palo Alto,
Calif.), or dual-luciferase reporter assay systems (Promega,
Madison, Wis.)]. The pGL3-enhancer parental vector was used as a
negative control and the pGL3-control vector with SV40 promoter was
used as a positive control for the luciferase activity. The
luciferase activity was counted on a luminescent light detector
such as a MicroBeta counter or a Luminometer (EG & G Life
Sciences-Wallac, Turku, Finland), and the result was normalized by
transfection efficiency. There was about a 5-fold increase of
luciferase activity for the human myostatin promoter construct in
comparison to its parental vector in human skeletal muscle cells
(FIG. 5A). In human rhabdomyosarcoma cells, the increase of
luciferase activity was about 7-fold (FIG. 5B). As expected, the
luciferase activity was not increased in human prostate smooth
muscle cells (FIG. 5A), which do not express myostatin.
[0038] In view of the above results, the luciferase reporter
construct containing the 3.4 kb human myostatin promoter region may
be stably or transiently transfected into mammalian cell lines such
as SV-40-transformed human skeletal muscle cells or
rhabdomyosarcoma cells and others for high through-put screening.
Luciferase activity may be used as an indicator for selecting
compounds which inhibit expression of the myostatin gene.
EXAMPLE III
Enzyme-linked Immunosorbent Assay (ELISA) for High Throughput
Screening
[0039] Sandwich or competitive ELISA may be used to identify
compounds which inhibit human myostatin protein synthesis and/or
secretion. In the sandwich ELISA, for example, a rabbit or mouse
monoclonal and/or polyclonal antibody against the mature form of
myostatin is coated on the Immulon-4 plate (Dynatech Laboratories,
Inc., Chantilly, Va.), and the plate is blotted by BSA, and washed.
Samples (e.g., supernatants from human skeletal muscle cells
treated with or without test agents) or known concentrations of
purified mature form of myostatin are added to the plate. After
myostatin protein binds, the plate is again washed, and then
incubated with a goat or rabbit or mouse monoclonal and/or
polyclonal antibody against myostatin. The binding of the second
anti-myostatin antibody will be detected by an antibody conjugated
with horseradish peroxidase (HRP) using 0-Phenylenediamine
hydrochloride (OPD) as the substrate. Known concentrations of
myostatin are used for generating the standard curve.
[0040] In the competitive ELISA, human myostatin protein is coated
on the Immulon-4 plate, and the plate is blotted by BSA, and
washed. Samples (e.g., supernatants from human skeletal muscle
cells treated with or without test agents) or known concentrations
of purified mature form of myostatin or myostatin peptides are
added to the plate along with a goat or rabbit or mouse monoclonal
and/or polyclonal antibody against myostatin. The plate is again
washed, and then incubated with an antibody conjugated with
horseradish peroxidase (HRP) using 0-Phenylenediamine hydrochloride
(OPD) as the substrate. The known concentrations of myostatin are
used for generating the standard curve.
[0041] Human skeletal muscle cells or other cell lines of muscle
lineage which synthesize and secrete myostatin may be used to test
compounds thought to inhibit the synthesis or secretion of
myostatin. Cells are incubated with test compounds for a period of
time, e.g., 6-48 hours. The amount of myostatin in the medium of
cells is then determined by ELISA. A decrease in the amount of
myostatin indicates that the test compound is effective in
inhibiting the synthesis and secretion of myostatin, whereas an
increase in the amount of myostatin or maintenance of the same
level of myostatin indicates that the test compound is not
effective in inhibiting the synthesis and secretion of
myostatin.
EXAMPLE IV
Production of Radiolabeled Myostatin for Use in Receptors Binding
Assay
[0042] The purified recombinant myostatin protein from bacteria,
insect or mammalian cells is radiolabeled ([.sup.125I], [.sup.3H],
[.sup.14C], etc.). The radiolabeled myostatin is then incubated
with cells or membranes prepared from tissues or cells which
contain myostatin receptors in the presence or absence of the test
composition. The amount of myostatin binding to membranes is
determined by counting radioactivity. A decrease in radioactivity
in the presence of a test composition indicates that the
composition inhibits myostatin binding, and thus is useful in
inhibiting myostatin function.
EXAMPLE V
Purified Myostatin Used in Affinity Selection
[0043] The purified recombinant myostatin protein from bacteria,
insect or mammalian cells is used in binding test compositions. In
particular, test compositions that bind to myostatin are retained
by filter, and the structure is determined by Mass
Spectophotometry. A test composition that binds to myostatin is
useful in inhibiting the binding of myostatin to its receptor, thus
inhibiting myostatin function.
Sequence CWU 1
1
3 1 3435 DNA Homo sapiens 1 ttctctaccc actcacccta atgatgcagt
actgtcctgt ctccttggtg ataagaactg 60 ccagaactgg gtctccagca
gtcagactac attgaagttt cctatagctg gtgagccctt 120 ctattctggg
gcctcaggaa ggttgcaatc actgccactg gagaaggaat aaaacttact 180
taaatttctt cagttcttct tcacccattc aatactgttc tctagagaag ttgattagat
240 atagtcaatt ctccctattc atagtagtta tgttctataa agtcattgcc
aacactgagt 300 tagcaaatat tgaactattg ttcccagggg aaaaacaggg
ttagttgagc ctctggtcat 360 aacatttaca tcacccaatc aatatataac
cttgttttat gtatgttttt gtttacaaat 420 acattattta atatatattg
ttcattcatt aacactgaac tcacagccag cagcactata 480 actcaggcct
gaatgatgct tatctagcac atgcattttt tcatgagaac ttttttccct 540
taggcatatc acagcctttt aaaattgtgg taaatataca caacatttag catcttaacc
600 atttttaggt gtacagttcg gtggcattaa gcacattcac actgttgtgt
aaccatcacc 660 accattcatc ttcagaaatt tttcatcttc ccagactgaa
actctgtatc tatcacacag 720 taactacccc tcagtgcctc acccagtccc
tggcaaccac catgctactt tccattgcag 780 ctttcctgtg attaggaaca
taggaacacc acgcagcacg tcagcactat gcttgggggc 840 catttttaaa
aagcaaaatc aataagagga gcaataaaaa aagaagcaca aaatatgtga 900
aaacatggca ctaaatagac gggaaaaagg gaatttgttt atagtatgag agctgaaaca
960 aaaaggcgga accctgcctt gtttgacctc atctgggaac ctgtgcgtca
gataggactc 1020 aaattttttg ccgctctgtg aatgccagcg aatgactgaa
aaatgctagg aatattgagt 1080 ttcatagata aattttagca agtaggcaaa
tttgtgaata cagaaatcat gaataataag 1140 aattgtctgt tagcggaaat
gacaaccttt gttcactttt ttccaggtga taagaataaa 1200 gagctggaac
atgtcttgta acctggctgc cttagaaaat tttacttgcc tcacaggcct 1260
agaaagtagg ctttgtgact gataattggc agctatgacc tgaagcagtt ctagttcatg
1320 tggagcataa ttttaagcat aatctcaaac ccttctgcat aaaacaaaga
gcaagcactc 1380 aaatgccagt tatcaattac ttactatatg acaggtgcca
tattcagcaa tttacatgca 1440 ttattaaatt atatcccccc aaaaccctat
gaggaagcta aagtttaggg aagttaagta 1500 tctcatccat tatcacatag
ttagaagtgg caaagttgag atttgaactc aggtctatct 1560 gactccagag
cctgagttct caattcaact gctatacaat tctaagcata ttaaaaaaaa 1620
agtttgactt acttggaact gtatagatgc atgtgttaca atgatcataa catttgaaag
1680 atttacacat tgaaaaatga atttaccaaa caaataaaac cttagaagcc
agatctaata 1740 ttgtcccata acaaagagta tctgaaatcc tcagggcatc
tggtttgtgt ctggttttcc 1800 ttaatcttta atgatgagca aatccaatgc
attatgtaag gccatttttt tctcaagaga 1860 tgtagatacc tcttaagaat
ttgatgaaaa tgcattaact tttcaggcta ctgagttgca 1920 ttttagtgca
ccgaggcagt aaattagtgt acagtgtgca aaaatggtag tgactttaaa 1980
aataaatatt tgatatgagc cactgtattc tcttggaaaa aaaaagtaat ggactaaatc
2040 tcttaggaat ccttagcttc ccaaaaagga gtaggaaaaa gaaatctcct
ttggcctaga 2100 aatatcttct gtttcttgct ggctatgttt gcttagctct
ttaatagttc atttgactag 2160 atcttgtggc tcccaaagct aaggttgaac
gtttgatccc tacagaggcc acttaaattt 2220 agagaacaaa aagctctatt
ctctgctccc agactttacc ccaaatccct gccaggtgtc 2280 tgccctctgg
tcaaaatgag aaattggcaa aggggtgcaa acatatcgca gtattgggaa 2340
acaacaaaag gtcacccctt tatcatgatg ctctttctct tttatgtgct cataatattc
2400 tgatataatt tatagagaat agatactgca ctttttactc tctggatatt
tactgctgga 2460 aatctgaggc aaactgtaat aatctctgcc atgccagtta
taaaattcat tatcttagtc 2520 tatgttcaga gatttttcta ctagctggca
ttaccctctt ggtaataaac aatgaaaaac 2580 acatcttctg agttatgtta
atctgcatct ttagaatagg aaataatagc actcagtcaa 2640 aagttcagta
taattttcat attaataaaa gacatgaaac tatgtaaaaa taattccatg 2700
cacaatatgt tataataaca atgacttcca atatttacta agaatttagt cagaaaacaa
2760 gtttctcaaa ttatagatga aaattctcaa ctagtatcat aatcttaact
tttaattcag 2820 gtcttcctaa tttttatttt cctaattact tggcactaaa
aataatttaa tacaacaaat 2880 aaaaatattt tctacttcaa atacttgcct
aaacaatata aaatcatttt agtttttgag 2940 gaagtaatat ttcatatttt
aaatatgtag tataaattaa aattgactta tttaaattac 3000 aataagagtt
gtgtgaggat tagtaagatt taagtacagt ttatattatt gccaacatag 3060
acttttgttt ttcaaatgtc acaaatatct tttattattt gtagatttat ttcttttatg
3120 aagtagtcaa atgaatcagc tcacccttga ctgtaacaaa atactgcttg
gtgacttggg 3180 acagacaggg ttttaacctc tgacagcgag attcattgtg
gagcaagagc caatcataga 3240 tcctgacgac acttgtctca tctaagttgg
aatataaaaa gccacttgga atacagtata 3300 aaagattcac tggtgtggca
agttgtctct cagactgtac atgcattaaa attttgcttg 3360 gcattactca
aaagcaaaag aaaagtaaaa ggaagaaaca agaacaagaa aaaagattat 3420
attgatttta aaatc 3435 2 25 DNA Homo sapiens 2 atgcaaaaac tgcaactctg
tgttt 25 3 21 DNA Homo sapiens 3 tcatgagcac ccacagcggt c 21
* * * * *