JP2003504351A - How to inhibit muscle atrophy - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for inhibiting atrophy in muscle cells. The invention further provides a method for inhibiting skeletal muscle atrophy or causing skeletal muscle hypertrophy in a vertebrate. The present invention also provides methods for identifying factors, genes and gene products that can be used to reduce proteolysis or increase protein synthesis in muscle cells. The present invention provides a method of inhibiting skeletal muscle atrophy by inhibiting a signaling pathway that causes skeletal muscle atrophy. Factors that inhibit Ras activation or that inhibit signaling molecules downstream of Ras (eg, Raf / Mek / Erk) prevent or reduce atrophy in skeletal muscle cells and / or cause hypertrophy. Useful in the invention.

Description

Detailed Description of the Invention

This application is US provisional application No. 60 / 142,857 (filed on July 7, 1999) (
This is incorporated herein by reference in its entirety).

BACKGROUND OF THE INVENTION Reduction or atrophy of muscle mass is associated with various physiological and pathological conditions. For example, muscular atrophy can result from nerve trauma; degenerative, metabolic or inflammatory neuropathy (eg, Guian-Barre syndrome); peripheral neuropathy; or denervation resulting from nerve damage caused by environmental toxins or drugs. . Muscle atrophy also refers to motor neuropathy (eg, adult motor neuron disease (eg, amyotrophic lateral sclerosis (AL)).
S or Lou Gehrig's disease); infantile and juvenile spinal muscular atrophy; and autoimmune motor neuropathy with multifocal conductor block)). Muscular atrophy can also result, for example, from paralysis resulting from stroke or spinal cord injury; skeletal fixation resulting from trauma (eg, fracture, ligament or tendon injury, sprain or dislocation); or from chronic illness resulting from long-term bed care. Can also occur. Metabolic stress or nutritional deficiencies, which can also result in muscle wasting, include cancer malignant fluids and other chronic illnesses, including AIDS, fasting or rhabdomyolysis, and endocrine disorders, among others. For example, thyroid disorders and diabetes mellitus). Muscular atrophy also refers to muscular dystrophy syndrome (eg Duchenne, Becker type, myotonic, fascioscapulohumera).
1), emery-de-lifes, oropharyngeal muscle, scapula, limb girdle and congenital type, and dystrophies known as hereditary distal myopathy). Muscle atrophy may also be due to congenital myopathy (eg, benign congenital hypotonia), central core disease, nemalene myopathy, and myotubular (central nucleus) myopathy. Muscle atrophy also occurs during the aging process.

Muscle atrophy at various pathological stages is associated with enhanced proteolysis and reduced production of muscle proteins. Muscle cells contain lysosomal and cytosolic proteases. Cytosolic proteases include Ca ²⁺ -activated neutral protease (calpain) and the ATP-dependent ubiquitin proteasome proteolytic system. Lysosomal and cytosolic systems can degrade muscle proteins in vitro, but little is known about their role in muscle protein degradation in vivo. Several studies have reported that proteasome inhibitors reduce proteolysis in atrophied rat skeletal muscle (eg, Tawa et al. (1997) J. Clin. Inve.
st. 100: 197), which suggests that the ubiquitin-proteasome pathway has a role in enhancing proteolysis. However, the exact mechanism of protein degradation in this atrophied muscle remains poorly characterized.

Ras is a signaling molecule found in all cells in the body.
It is activated by various growth factors, including those that signal tyrosine kinase receptors. Ras activation results in the activation of several downstream signaling pathways, including the Raf / Mek / Erk pathway. R
Other potential downstream substrates of as include the following signaling molecules:
PKC, Ral-GDS, KSR and Rin-1 (Trends Genet April 1999; 15 (4): 145-9). The Ras, Raf, Mek and Erk genes are well conserved throughout evolution, and homologues can be found in eukaryotic cells from yeast to human (Ras: Taparowsky E et al. (1982) Nature 300: 762-). 5; Raf: Heidec
ker et al. (1990) Mol. Cell. Biol. 10: 2503; Samu
els et al. (1993) Mol. Cell. Biol. 13: 6241; Morr
ison et al. (1997) Curr. Opin. Cell Biol. 9: 174
Mek: Crews CM et al. (1992) Science 258: 478-.
80; Erk: Boulton TG et al. (1991) Cell 65: 663-.
75).

When a constitutively active mutant of Ras was expressed in the heart muscle of transgenic mice, this animal was born with hypertrophic heart tissue (J Biol).
． Chem. 1995 Sep 29; 270 (39): 23173-8). This suggests that Ras activation may cause hypertrophy in skeletal muscle as well, and shows that Ras activation may be one effective way to restore skeletal muscle atrophy.

However, it has been disclosed according to the invention that proteolytic or diminished synthesis in muscle cells is indeed increased by activation of Ras and cell signaling molecules in the downstream Raf / Mek / Erk pathway.

SUMMARY OF THE INVENTION The present invention provides for the inhibition of signaling pathways that cause skeletal muscle atrophy,
A method of inhibiting skeletal muscle atrophy is provided. Factors that inhibit Ras activation or Ra
Factors that inhibit signaling molecules downstream of s (eg Raf / Mek / Erk) are useful in the invention to prevent or reduce atrophy and / or cause hypertrophy in skeletal muscle cells.

The transgene, transfected ES cells are injected into the host blastocyst and further into the pseudopregnant foster host, followed by embryonic development and the birth of transgenic mice. A constitutively active mutant form of a Ras, Raf, Mek or Erk nucleic acid is expressed in muscle cells of transgenic mice and is a constitutively active mutant form of Ras protein, Raf protein, Mek protein or E.
Provides rk protein to muscle cells.

In another preferred embodiment, transgenic mice are made by injecting a vector containing the transgene directly into the pronucleus of fertilized eggs, as described in Hogan et al. (Supra). The injected eggs are inserted into a pseudopregnant foster mother host, followed by embryonic development and the birth of transgenic mice.

The presence of constitutively active mutant forms of Ras, Raf, Mek, or Erk proteins has also been demonstrated using antibodies specific for these proteins or in vectors encoding these proteins. Can be assessed by an antibody specific for the epitope tag genetically inserted in.

In a method for the identification of a factor that inhibits myocyte atrophy, the factor is a muscle cell that expresses a constitutively active mutant form of Ras, Raf, Mek or Erk by methods known in the art. Can be contacted with. For cells in culture or obtained from transgenic organisms, these cells can be contacted with the factor by, for example, direct application. This factor may be modified or included in the delivery vehicle to facilitate entry into the cell. This factor may be isolated and purified, or may be present in a sample or composition subject to further purification following positive results in the method. For example, the reagent can be contained within a cell. Skeletal muscle cells have Mek kinases (which are Ras and R
It was discovered according to the invention that the resulting cells appear hypertrophic when compared to control cells when treated with chemicals that block signaling (downstream of af).

Accordingly, the Applicants have determined that Ras or its downstream signaling molecule Raf / Me.
It has been discovered that inhibitors of k / Erk are useful for reducing or preventing atrophy in skeletal muscle cells. Further, such inhibitors can be used to reduce and / or prevent atrophy in mammals having conditions as described herein, in which skeletal muscle atrophy occurs. In a preferred embodiment,
Such inhibitors are pharmacological antagonists for Ras [Can
cer Chemother Pharmacol. (1999) 43 (1):
50-8], or inhibitors already known to be pharmacological antagonists for Raf / Mek / Erk (eg PD98059.
[Proc. Natl. Acad. Sci. (1995) 92: 7686] or farnesyl transferase (Chem. Biol (1995) Dec.
2 (12): 787-91)). According to this embodiment, skeletal muscle cells undergoing atrophy, or vertebrates having a condition such as those in which the muscle cells are undergoing atrophy, are treated with an inhibitor to prevent or reduce myocyte atrophy. Such treatment may be used prophylactically before the onset of muscular atrophy, or after the appearance of such a condition. Vertebrate animals include any species including skeletal muscle and skeleton, including chickens, rodents, rabbits, dogs, cats, cows, horses, pigs, goats, primates and humans, preferably Is a human.

In another embodiment, Ras or the downstream signaling molecule Raf /
Inhibitors of Mek / Erk can be used to effect atrophy in skeletal muscle cells. Further, such inhibitors can be used to effect muscle atrophy in vertebrates having conditions as described herein in which skeletal muscle atrophy is predicted. In some settings, such as animals raised for meat production, such factors can be used to increase meat production. In a preferred embodiment, such inhibitors are pharmacological antagonists for Ras [Cancer Chemother Pharmacol. (199
9) 43 (1): 50-8], or inhibitors already known to be pharmacological antagonists for Raf / Mek / Erk (eg,
PD98059 [Proc. Natl. Acad. Sci. (1995) 92:
7686] or farnesyl transferase (Chem. Biol (19
95) Dec 2 (12): 787-91)). According to this embodiment,
Skeletal muscle cells undergoing atrophy, or vertebrates with conditions such as those in which muscle cells are undergoing atrophy, are treated with an inhibitor to prevent or reduce muscle cell atrophy as a result. Such treatments may be used prophylactically before the onset of muscle wasting, or after the appearance of such conditions. Alternatively, such treatment can be used to induce skeletal muscle hypertrophy.

The present invention further provides a Ras antagonist or Raf / Mek / Erk in a carrier, which may include excipients, diluents or other pharmaceutically active compounds.
Provided are therapeutic compositions that include an antagonist. Such compositions can be administered systemically or locally. Any suitable dosage form known in the art may be used, including intravenous injection, intrathecal injection, intraarterial injection, intranasal injection, oral injection, subcutaneous injection, intraperitoneal injection, or local injection. , Or, but not limited to, surgical implantation. Sustained-release formulations are also provided.

The activity of the compositions of the invention in vertebrates can be evaluated using experimental animal models of disorders in which muscular atrophy is present. For example, the activity of this composition can be tested in Example 2 for their effect on the hindlimb fixation model described herein. Alternatively, the activity of this composition can be evaluated using laboratory animals in which hypertrophy can be measured. For example, the activity of this composition may be due to exercise-induced hypertrophy.
-Induced hypertrophy or compensation-induced hypertrophy
can be tested for their effect on muscle undergoing nation-induced hypertrophy. Alternatively, muscle can be evaluated in control animals compared to animals treated with the experimental composition to determine whether the treated animals exhibit skeletal muscle hypertrophy as a result of the treatment. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in vertebrates, including humans. The dosage of the compositions herein should be within a range of circulating serum concentrations that have little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration.

[0016] In another embodiment, a method of identifying a factor that inhibits atrophy or causes hypertrophy in muscle cells is provided. The method is Ras or Raf / Mek /
Generating a myocyte expressing a constitutively active variant of Erk, contacting this myocyte with a factor to be tested; and a cell that has not undergone atrophy or the amount of atrophy is compared to a control cell. Screening for cells that have been reduced.

In one embodiment, the invention provides a method of identifying a factor that inhibits atrophy or causes hypertrophy in muscle cells. This method comprises preparing an in vitro assay for Ras activity or Raf / Mek / Erk activity,
Contacting Ras or Raf / Mek / Erk with the agent being tested; and screening for agents that block Ras activity or Raf / Mek / Erk activity.

Another preferred embodiment of the invention provides a method of identifying a gene encoding a gene product that inhibits atrophy or causes hypertrophy in muscle cells. This method comprises the step of preparing muscle cells expressing a constitutively active mutant of Ras or Raf / Mek / Erk; the genes to be tested in these muscle cells under the conditions that the test gene encodes a product. And the step of measuring the amount of atrophy in the muscle cells encoding this test gene, and the amount of atrophy in the cells encoding this test gene are compared with the amount of atrophy in the muscle cells into which the test gene has not been introduced. The step (wherein a smaller amount of atrophy in the muscle cells encoding the test gene results in the test gene product inhibiting the Ras pathway or the Raf / Mek / Erk pathway, and therefore in cells under conditions where such atrophy occurs. Indicates that it inhibits atrophy)
Includes.

Cells useful for expressing constitutively active mutant forms of Ras or Raf / Mek / Erk can be maintained in culture and engineered to express a heterologous nucleic acid, any Includes myocytes and all myocytes. These cells can be primary cultures or established cell lines. Suitable muscle cells include myoblasts such as those described by Bains et al. (1984) Mol. Cell. Biol. 4: 1449-15
53, the disclosure of which is incorporated herein by reference, and includes the C2C12 cell line. Other suitable muscle cells include Glass et al. (1997) Proc. Natl. Acad. Sci. USA 16: 8848
Sol8 cells described in, and Ringentz et al. (1978) Exp. C
ell. Res. L6 cells as described in 113: 233, the disclosures of which are incorporated herein by reference.

Cells useful for making cultured muscle cells expressing the Ras gene or the Raf / Mek / Erk gene also include non-muscle cells that can differentiate into skeletal muscle cells; examples of this cell type. Are embryonic stem cells (which are described in Shani et al. (1992)
) Symp. Soc. Exp. Biol. 46:19, it can differentiate into skeletal muscle cells by insertion of the muscle-specific transcription factor MyoD).
Another example of non-muscle cells that can be differentiated along the skeletal muscle pathway is rhabdomyosarcoma tumor cells, which are described in Gabbert et al. (1988) Cancer Res. 48
: 5264 and may be differentiated by contacting with retinoic acid.

Ras or Raf under the control of appropriate transcriptional and translational regulatory sequences
The nucleic acid in the constitutively active mutant form of / Mek / Erk can be prepared by methods known in the art (
(Including, for example, transformation, transfection, infection, transduction, and infusion). In a preferred embodiment, Ras gene, Raf
The gene, Mek gene, or Erk gene is included in the expression vector under the control of a suitable promoter that drives expression in muscle cells. Preferred promoters include human skeletal actin (HSA) and chicken skeletal actin (CSA)
, Skeletal actin such as mouse skeletal actin (MSA), rat skeletal actin (RSA); muscle creatine kinase (MCK); MyoD; MRF4; myogenin; dystrophin; utrophin; MuSK; myosin light chain (MLC1 / 3)
The myosin heavy chain (MHC). Other non-muscle-specific promoters include cytomegalovirus (CMV); LTR (long terminal repeats, found in retroviruses including Moloney murine leukemia virus (MuLV)).
); HTLV-1 (promoter of human T cell leukemia virus 1); late (la
te) adenovirus, middle adenovirus, or early (e)
aly) adenovirus.

The amount of atrophy in cells can be measured by quantification of diameter, quantification of protein content, or by activation of p70S6 kinase or Phas-1 protein that stimulates protein synthesis. In transgenic organisms, muscle wasting can be measured as described in Example 2 herein.

In yet another preferred embodiment, Ras, Raf, Mek, or Erk proteins are expressed in an in vitro assay so that agents that inhibit these signaling molecules can be screened. Alternatively, an in vitro binding assay is utilized to utilize Ras, Raf, Me
Agents that block the binding of k, or Erk, to downstream signaling components can be identified. This is described, for example, by End et al. biol. Chem.
268 (14): 10066-10075.

Expression vectors containing a nucleic acid of a constitutively active mutant form of Ras, Raf, Mek or Erk under the control of a suitable promoter can be obtained by known methods (eg, liposome-mediated transfection, calcium phosphate-mediated transfection, D
EAE-dextran transfection, transfection of naked DNA, microinjection, electroporation, retrovirus-mediated infection, adenovirus-mediated infection, or adeno-associated virus-mediated infection). The constitutively active mutant form of nucleic acid of Ras, Raf, Mek or Erk can be introduced into cells in a stable or transient manner. Methods for introducing heterologous nucleic acids into eukaryotic cells are described in numerous laboratory manuals (eg, DNA Clon).
ing: A Practical Approach, Volumes I-III (198)
5) Glover et al., IRL Press Limited, Oxford, and Sambrook et al. (1989) Molecular Cloning: A.
Laboratory Manual, 2nd Edition, Cold Spring H
Arbor, NY.

In a preferred embodiment, constitutively active mutant forms of nucleic acid of Ras, Raf, Mek or Erk are described, for example, in Pear et al. (1993) Proc. Natl
． Acad. Sci. USA 90: 8392 (incorporated herein by reference) and inserted into a retroviral vector. In this embodiment, the viral LTR promoter controls a constitutively active mutant form of nucleic acid of Ras, Raf, Mek or Erk. This vector was transiently transfected into and produced a retroviral packaging strain,
Recombinant viruses containing constitutively active mutant forms of nucleic acids of Ras, Raf, Mek, or Erk are harvested as described by Pear et al. (Id.).
The recombinant virus was then cloned into Hoffman et al. (1996) Proc. Natl
． Acad. Sci. USA 93: 5185 (incorporated herein by reference) and used to infect myoblasts. As an example of skeletal muscle cells, C2C12 cells expressing a constitutively active mutant form of Ras, Raf, Mek, or Erk are undifferentiated by growing them in tissue culture medium containing at least 10% fetal calf serum. Or can be differentiated into skeletal muscle myotubes by growing in medium containing 2% horse serum. The required tissue culture methods are known to those of skill in the art. C2C12 cells were obtained from Bains et al. (1
984) Mol. Cell Biol. 4: 1449 (incorporated herein by reference).

Muscle cells expressing a constitutively active mutant form of a nucleic acid of Ras, Raf, Mek or Erk can be contained within or obtained from a transgenic organism. As used herein, the term transgenic organisms refers to foreign genes (especially Ras, integrated into their genome).
A myocyte-containing multicellular organism having a constitutively active mutant form of Raf, Mek or Erk). Transgenic organisms contemplated according to the present invention include helminths, birds, chickens, turkeys, flies, and non-human mammals such as mice, rats, dogs, rabbits, sheep, pigs, sheep, goats, horses and cows. )including. Transgenic helminths and transgenic mice are particularly preferred for research use according to the invention.

In the transgenic organism of the invention, R under the control of muscle-specific regulators
The coding region for a constitutively active mutant form of an as, Raf, Mek or Erk nucleic acid is integrated into the genome of the host organism. Muscle-specific regulators may be Ras, Raf, Mek or Er to provide for expression of these nucleic acids in muscle cells of the host organism.
It is directed to the expression of a constitutively active mutant form of a k nucleic acid and can be native to the host organism or heterologous to the host organism. Preferred muscle-specific regulators useful in the present invention are selected for optimal expression in a particular host. Preferred muscle-specific regulators for use in non-human transgenic mammals include the human skeletal actin (HSA) promoter (Brennan et al.
1993) J. Biol. Chem. 268: 719, incorporated herein by reference) and muscle keratin kinase (MCK) promoter (Johnso).
n et al. (1989) Mol. Cell. Biol. 9: 3393, incorporated herein by reference).

The transgenic organism of the invention comprises a transgene DNA comprising a coding region for a constitutively active mutant form of a Ras, Raf, Mek or Erk nucleic acid operably linked to a muscle-specific promoter entering the host organism. Produced by introducing the construct so that the transgene is integrated into the genome of the host organism. Methods for producing transgenic non-human organisms are known in the art and include, for example, DNA microinjection, embryonic stem (ES) cell transfer, retroviral infection, blastomere agglutination, teratocarcinoma cell transfer, Electrofusion, nuclear transfer, and sperm-mediated transfer are included. The method is described, for example, in Pinkert et al., “Transge
nic Animal Modling ", Molecular Biolog
y and Biotechnology, Meyers, VCH Publ
ishers, Inc, New York, 1995, pp. 90-907, and
For example, Hogan et al. (1994) Manipulating the Mou.
se Embryo; A Laboratory Manual, Second Edition, Co
ld Spring Harbor Laboratory Press, Pl
It is reviewed by numerous laboratory manuals, including ainview, New York, the disclosures of which are incorporated herein by reference. In a preferred embodiment of the invention, transgenic non-human organisms are produced by microinjection. Transgenic organisms contemplated according to the present invention include transgenic flies, helminths, birds, chickens, turkeys, mice,
Examples include rats, dogs, cats, rabbits, sheep, pigs, goats or horses,
It is not limited to these. In a preferred embodiment of the invention, the transgenic organism is a transgenic mouse. Methods for making transgenic helminths, particularly Caenorhabditis elegans, are also known in the art, and are described, for example, in Mello et al. (1991) EMBO. J. 10:
3959.

In a preferred embodiment of the invention, the transgenic organism is a transgenic mouse, the germ cells and somatic cells of which are Ras, Raf, Mek or Erk nucleic acids operably linked to a muscle-specific promoter. A transgene containing a constitutively active mutant form of the coding region, whereby Ras, Raf,
A constitutively active mutant form of the Mek or Erk protein is thus produced in muscle cells of transgenic organisms. The transgenic mouse preferably transfects ES cells using a vector containing the transgene, injects the transfected ES cells into a host blastocyst, and further into a pseudopregnant mother-in-law host, It is subsequently created by embryonic development and the birth of transgenic mice. A constitutively active mutant form of a Ras, Raf, Mek or Erk nucleic acid is expressed in muscle cells of a transgenic mouse and provides a constitutively active mutant form of Ras, Raf, Mek or Erk protein in the muscle cell. To do.

In another preferred embodiment, transgenic mice are produced by directly injecting a vector containing the transgene into the pronucleus of fertilized eggs as described by Hogan et al. (Supra). . The injected eggs are inserted into a pseudopregnant mother-in-law host, followed by embryonic development and the birth of transgenic mice.

The presence of constitutively active mutant forms of the Ras, Raf, Mek or Erk proteins is also genetically determined using antibodies specific for these proteins or in the vectors encoding these proteins. It can be evaluated by an antibody specific for the epitope tag inserted into.

In a method for the identification of a factor that inhibits myocyte atrophy, the factor is a muscle expressing constitutively active mutant form of Ras, Raf, Mek or Erk, according to methods known in the art. It can be contacted with cells. For cells in culture or obtained from a transgenic organism, the cells can be contacted with the factor (
For example, by direct application). The factor, in order to facilitate entry into the cell,
It may be modified or included in the delivery vehicle. The factor may be isolated and purified, or may be present in a sample or composition subject to further purification following the positive results of the method. For example, the factor can be included in a cell lysate, conditioned cell culture medium, or a library of synthetic or naturally occurring compounds. For muscle cells present in transgenic organisms, the cells may be prepared by methods known in the art (eg, oral ingestion, parenteral administration,
Alternatively, the agent can be contacted with it by delivery (by direct application to the tissue surface) and can be present in a composition containing a carrier or diluent. Agents that may be tested in the methods of the invention include, for example, organic and inorganic molecules such as proteins, peptides, lipids, carbohydrates, nucleic acids, including antisense, metals, salts and the like.

In the method of identifying a gene, the gene product of which inhibits myocyte atrophy in the presence of a constitutively active mutant form of Ras, Raf, Mek or Erk, the gene tested is , Ras, Raf, Mek or Erk signaling efficiency can be introduced into muscle cells by methods known in the art and described herein above. The gene may be under the control of a promoter and may be in an expression vector, as described herein. Pools of genes (eg, cDNA libraries) can be tested for their ability to interfere with proteolysis. Upon identification of gene pools that inhibit proteolysis, the pools can be subdivided and tested until a single gene is identified.

In a method of identifying a gene, the expression of which inhibits atrophy in muscle cells containing a constitutively active mutant form of Ras, Raf, Mek or Erk, the genetic mutation is known in the art. It can be introduced into a gene in muscle cells by a method. Directed mutagenesis methods are known in the art and are described, for example, in Directed.
Mutagenesis: A Practical Approach (199
1) Edited by McPherson, Oxford University Press
, Oxford, can be found in the laboratory manuals. In a preferred embodiment, random mutagenesis is accomplished using the chemical entity EMS, which can be accomplished by one of ordinary skill in the art. One example can be found in Chanal et al. (1997) Genetics 146: 20. The method of inducing mutation is
son (1995) Methods Cell Biol. 48:31.

The method of the invention is useful for the identification of factors, genes and gene products that interfere with atrophy in myocytes containing constitutively active mutant forms of Ras, Raf, Mek or Erk. The factors, genes and gene products identified by this method are useful for the treatment and prevention of muscular atrophy.

[0036]   The references cited herein are incorporated herein in their entirety.

[0037]   The following non-limiting examples serve to further illustrate the present invention.

(Example) (Example 1) (Effect of Raf activation and blocking in muscle cells) To test the effect of activating and blocking Raf in muscle cells,
The myoblast cell line called C2C12 has a constitutively active Raf gene [Heide]
cker, et al. (1990) Mol. Cell. Biol. 10:25
03; Samuels, et al. (1993) Mol. Cell. Bio
l. 13: 6241; Morrison, et al. (1997) Curr
． Opin. Cell. Biol. 9: 174] or the dominant negative Raf gene [Bruder, et al. (1992) Genes Dev.
6: 545], or as a control, a DNA expression vector containing an expression vector without any gene inserted. A "dominant negative" construct may block the activity of an endogenous gene product;
A "constantly active" construct encodes a mutated form of a gene that is always in the activated state. These constructs were made using standard molecular biology techniques and then stably transfected into C2C12 cells using the calcium phosphate transfection method. Transfected cells were selected by antibiotic selection. Then, the obtained myoblasts were grown to confluence and differentiated into “myotubes”, which are multinucleated myocytes similar to actual myofibers.

It was found that the myotubes expressing the dominant negative form of Raf are significantly longer than the control myotubes-the myotubes are longer and have a wider diameter. Phas-1, a molecule involved in protein synthesis [Xu, e
t al. (1998) J. Biol. Chem. 273 (8): 4485-4.
491] analysis revealed that inhibition of Raf causes an increase in Phas-1 activation. Thus, blocking Raf was shown to increase muscle protein synthesis.

The myotubes expressing the constitutively active form of Raf were found to be considerably thinner than the control myotubes. By analysis of Phas-1 in these myotubes, Raf
Was found to cause a decrease in Phas-1 activation,
It was revealed that Raf activation reduces muscle protein mass and inhibition of Raf can be used to reduce or prevent muscle atrophy.

Furthermore, the myotubes of C2C12 were attached to PD98059 [Dudley, et al. (
1995) Proc. Natl. Acad. Sci. , 92: 7686], and compared with differentiated cells expressing dominant negative Raf. Cells that had been post-differentiated with Mek inhibitor 3 mM for one day were indistinguishable from dominant negative Raf cells.

Example 2 Animal Model for Testing for Muscle Atrophy To test for muscle atrophy, the ankle joint of a rodent animal (mouse or rat) is fixed at 90 ° flexion. This procedure causes atrophy of the muscles that move the ankle joints at various angles (eg, soleus, medial and lateral gastrocnemius, anterior tibialis).
The amount of reproducible atrophy can be measured in the hindlimb muscle over 14 days.

The fixation procedure may include cast plastering (mouse) or pinning (rat) of the ankle joint. The rodent is anesthetized with ketamine / xylazine and the right ankle is fixed. In the rat, a 0.5 cm incision is made in the heel area along the paw axis. A screw (1.2 x 8 mm) is then inserted through the calcaneus and tail into the long shaft of the tibia. The wound is closed with skin glue. In mice, the ankle joint is fixed at 90 ° around the joint with a lightweight cast bandage (VET-LITE). Soak the material in water and then wrap it around the limb. The material is hard when dry, but light in weight.

At 7 and 14 days post fixation, animals are anesthetized and killed by cervical dislocation. The anterior tibial muscle (TA), medial gastrocnemius (MG), and soleus (Sol) muscles (
Take from right (fixed) and left hindlimbs (intact), weigh and freeze in liquid nitrogen cooled isopentane at fixed length. A cohort of control animals of the same weight and age as this experimental animal is also killed, muscles removed, weighed and frozen. The amount of atrophy is evaluated by comparing the muscle weight from the fixed limb with the muscle weight from the control animal.

Example 3 Preparation of Muscle-Specific Eukaryotic Expression Constructs for Homeostatically Active Ras and Homeostatically Active Raf Homeostatically Active Ras-Encoding DNA and Homeostatic The DNA encoding Raf active in Raf was cloned into a eukaryotic expression vector by standard molecular biology techniques. Briefly, a DN encoding a constitutively active RasV12
DNA encoding the COOH termini (RafCT) of A and Raf, which is constitutively active when expressed, was gel purified and purified from muscle creatine kinase (MC).
K) cloned into a eukaryotic expression vector containing a promoter. These vectors were called MCK RasV12 and MCK RafCT, respectively. MCK
Promoters have previously been shown to cause muscle-specific expression of transgenes in mice [Johnson et al. (1989) Mol. Cell
． Biol. 9: 3393].

Example 4 Transfection of C2C12 Cells Mouse C2C12 myoblasts were used as a method to study transgenes in cell culture. To effect Ras and Raf gene expression, a vector using the mouse muscle creatine kinase (MCK) gene promoter was transfected into C2C12 by calcium phosphate precipitation as described in Example 3. To allow positive selection of transgenic C2C12 cells, the drug G418 (Gibco) was used to cotransfect the MCK-RasV12 and RafCt vectors with a plasmid containing the LTR gene promoter driving the neo gene. Expression of the plasmid allows the cells to resist the killing effect of the drug G418; thus, treatment with G418 only survives the successfully transfected cells.

Transfected C2C12 cell colonies were harvested, trypsinized and replated and grown in individual wells of 24-well culture plates. Control C2C12 cells, C2C12 cells with MCK RasV12 construct and C2C12 cells with MCK RafCT construct were expanded to confluency. Serum levels were reduced and myoblast fusion was performed to differentiated myocytes. C2C2C12 cells differentiated into multinucleated myotubes. The resulting myotube phenotype was quantified by diameter, protein content and activation of Phas-1 protein that stimulates protein synthesis.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 111 C12Q 1/02 C12N 5/10 1/68 Z 15/09 C12N 15/00 A C12Q 1 / 02 5/00 B 1/68 A61K 37/52 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC , NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, A , BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO , NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW ( 72) Inventor Lommel, Christian C.H. Switzerland-1227 Geneva, Les Acacias, Root des Acacias, 4, Studio House (72) Inventor Janko Paulus, George Dee. United States New York 10598, Yorktown Heights, Babistist Church Road 1519 F-term (reference) 4B024 AA01 AA11 BA80 CA02 DA02 EA04 FA02 HA11 4B063 QA05 QA08 QQ21 QQ41 QQ43 QQ61 QQ79 QQ89 QR77 QR80 QS31 QS38 QX10 4B050 BA02A4A14BAA01 BAB01BAA01 AB90A02. CA46 4C084 AA02 AA17 BA44 DC25 NA14 ZA94 ZB21

Claims (41)

[Claims] 1. A method of inhibiting atrophy in skeletal muscle cells, comprising treating the cells with an inhibitor of the Ras / Raf / Mek / Erk pathway. 2. The method of claim 1, wherein the inhibitor inhibits Ras. 3. The method of claim 1, wherein the inhibitor inhibits Raf. 4. The method of claim 1, wherein the inhibitor inhibits Mek. 5. The method of claim 1, wherein the inhibitor inhibits Erk. 6. The method of claim 1, wherein the inhibitor is PD98059 or farnesyl transferase. 7. A method for identifying a factor that inhibits atrophy in skeletal muscle cells, which comprises: (a) preparing a muscle cell that expresses a constitutively active mutant form of Ras / Raf / Mek / Erk. (B) subjecting the cells to a test factor; (c) measuring the amount of atrophy in the muscle cells subject to the test factor; (d) atrophy in the muscle cells subject to the test factor. And the amount of atrophy in the untreated transgenic myocytes of step (a), wherein:
Less atrophy in the myocytes subjected to the test factor is due to the fact that the factor is Ras / R
Inhibiting the af / Mek / Erk pathway and thus inhibiting atrophy in myocytes. 8. The measuring step comprises myocyte diameter, protein amount, p70S
The method according to claim 7, which utilizes 6-kinase activation or Phas-1 activation. 9. The method according to claim 7, wherein the measuring step utilizes measuring inhibition of Ras / Raf / Mek / Erk. 10. The method of claim 7, wherein the muscle cells are cultured cells. 11. The method according to claim 10, wherein the cultured cells are myoblasts. 12. The method according to claim 11, wherein the myoblast is a C2C12 cell. 13. The method of claim 11, wherein the myoblasts are differentiated myoblasts. 14. The method of claim 13, wherein the differentiated myoblast is a myotube. 15. The method of claim 7, wherein the muscle cells are obtained from a transgenic organism. 16. The muscle cell is in a transgenic organism.
The method described in. 17. The transgenic organism according to claim 15, which is a transgenic fly, helminth, bird, chicken, turkey, mouse, rat, dog, cat, rabbit, sheep, pig, goat or horse. Method. 18. The transgenic organism of claim 16, wherein the transgenic organism is a transgenic fly, worm, bird, chicken, turkey, mouse, rat, dog, cat, rabbit, sheep, pig, goat or horse. Method. 19. A method for identifying a factor that inhibits atrophy in muscle cells, comprising the steps of: a) measuring the activity of the Ras / Raf / Mek / Erk pathway in untreated muscle cells; b) Ras / Raf. Subjecting myocytes expressing the / Mek / Erk pathway to a test factor, c) measuring the amount of Ras / Raf / Mek / Erk activity in the myocytes subject to the test factor; d) subjecting to the test factor Comparing the amount of Ras / Raf / Mek / Erk activity in the myocytes treated with the amount in untreated myocytes, whereby greater amount in the myocytes treated with the test factor is , The factor is Ras / Raf /
Demonstrating inhibiting the Mek / Erk pathway and thus inhibiting atrophy in myocytes. 20. A method for identifying a gene encoding a gene product that inhibits skeletal muscle atrophy, comprising: (a) a muscle expressing a constitutively active mutant form of Ras / Raf / Mek / Erk. Preparing the cells; (b) introducing the test gene into the cells of (a) under conditions in which the test gene encodes a product; (c) determining the amount of atrophy in the muscle cells encoding the test gene. A step of measuring; and (d) a step of comparing the amount of atrophy in cells encoding the test gene with the amount of atrophy in muscle cells into which the test gene of step (a) has not been introduced,
Here, a lesser amount of atrophy in muscle cells encoding the test gene indicates that the test gene product inhibits the Ras / Raf / Mek / Erk pathway and thus inhibits atrophy in muscle cells. Including the method. 21. The measuring step comprises muscle cell diameter, protein amount, p70
21. The method of claim 20, which utilizes S6 kinase activation or Phas-1 activation. 22. The method of claim 20, wherein the muscle cells are cultured cells. 23. The method of claim 22, wherein the cultured cells are myoblasts. 24. The method of claim 23, wherein the myoblasts are differentiated myoblasts. 25. The method of claim 20, wherein the muscle cells are obtained from a transgenic organism. 26. The method of claim 2, wherein the muscle cells are in a transgenic organism.
The method described in 0. 27. The method according to claim 25, wherein the transgenic organism is a transgenic fly, helminth, bird, chicken, turkey, mouse, rat, dog, cat, rabbit, sheep, pig, goat or horse. Method. 28. The transgenic organism according to claim 26, which is a transgenic fly, helminth, bird, chicken, turkey, mouse, rat, dog, cat, rabbit, sheep, pig, goat or horse. Method. 29. A method of inhibiting atrophy in a vertebrate having an atrophy-inducing condition, comprising treating the vertebrate with an effective amount of an inhibitor of Ras, Raf, Mek or Erk. . 30. The method of claim 29, wherein the vertebrate is a chicken, rodent, rabbit, dog, cat, cow, horse, pig, sheep, primate or human. 31. The method of claim 29, wherein the vertebrate is treated prior to exposure to or development of the atrophy-inducing condition. 32. The method of claim 29, wherein the atrophy-inducing condition is fixed. 33. The method of claim 29, wherein the atrophy-induced condition is denervation, starvation, nutritional deficiency, metabolic stress, diabetes, aging, muscular dystrophy or myopathy. 34. A method of causing muscle hypertrophy in skeletal muscle cells comprising treating the cells with an inhibitor of the Ras / Raf / Mek / Erk pathway. 35. The method of claim 34, wherein the inhibitor inhibits Ras. 36. The method of claim 34, wherein the inhibitor inhibits Raf. 37. The method of claim 34, wherein the inhibitor inhibits Mek. 38. The method of claim 34, wherein the inhibitor inhibits Erk. 39. The method of claim 34, wherein the inhibitor is PD98059 or farnesyl transferase. 40. The method of claim 34, wherein the muscle cell is in a vertebrate. 41. The method of claim 40, wherein the vertebrate is a chicken, rodent, rabbit, dog, cat, cow, horse, pig, sheep, primate or human.