Screening assay employing Dex and GDF8
Certain aspects of this disclosure relate to a method that comprises contacting a mammalian cell with a glucocorticoid receptor ligand and a myostatin receptor ligand, thereby activating the glucocorticoid receptor and said myostatin receptor. A screening assay employing the same is also provided.
1. A method comprising:
contacting a mammalian cell comprising a glucocorticoid receptor, a myostatin receptor and a recombinant nucleic acid comprising an atrogen promoter that is at least 95% identical to a wild type MURF-1 or MAFbx promoter and operably linked to a coding sequence encoding a reporter protein, with a ligand that activates the glucocorticoid receptor and a ligand that is at least 90% identical to a wild-type mammalian GDF8 and activates the myostatin receptor, thereby activating said atrogen promoter and inducing expression of said reporter protein.
2. The method of claim 1 , wherein said contacting initiates an atrophy response by said mammalian cell.
3. The method of claim 1 , wherein said contacting is done by contacting said ligand that activates the glucocorticoid receptor and said ligand that activates the myostatin receptor with a cultured cell in vitro.
4. The method of claim 3 , wherein said ligand that activates the glucocorticoid receptor is contacted with said mammalian cell at a concentration in the range of 0.1 μM to 100 μM.
5. The method of claim 3 , wherein said ligand that activates the myostatin receptor is contacted with said mammalian cell at a concentration of 1 ng/mL to 1000 ng/mL.
6. The method of claim 1 , wherein said contacting is done by administering said ligand that activates the glucocorticoid receptor and said ligand that activates the myostatin receptor to a mammal.
7. The method of claim 1 , wherein said ligand that activates the glucocorticoid receptor and said ligand that activates the myostatin receptor are contacted with said mammalian cell simultaneously.
8. The method of claim 1 , wherein said ligand that activates the glucocorticoid receptor and said ligand that activates the myostatin receptor are contacted with said mammalian cell at different times.
9. The method of claim 1 , wherein said ligand that activates the glucocorticoid receptor is dexamethasone.
10. The method of claim 1 , wherein said ligand that activates the myostatin receptor comprises an amino acid sequence that is at least 95% identical to a wild-type GDF8.
11. The method of claim 1 , wherein said ligand that activates the myostatin receptor is GDF8.
12. The method of claim 1 , wherein said atrogen promoter is identical to a wild type MURF-1 or MAFbx promoter.
13. The method of claim 1 , wherein the contacting is done in the presence of a candidate agent; and the method further comprises measuring production of the reporter protein.
14. The method of claim 13 , wherein the method further comprises determining if said candidate agent alters cell phenotype of said cell.
15. The method of claim 14 , wherein said cell phenotype is a muscle atrophy phenotype.
16. The method of claim 13 , wherein said contacting is done by contacting said candidate agent with a cultured cell in vitro; and the method further comprises determining a muscle atrophy phenotype in the cultured cell.
17. The method claim 13 , wherein said contacting is done by administering said candidate agent to a mammal; and the method further comprises determining a muscle atrophy phenotype in muscle tissue of the mammal.
18. The method of claim 17 , wherein said determining a muscle atrophy phenotype comprises measuring muscle mass.
19. The method of claim 17 , wherein said mammal is a rat.
20. The method of claim 13 , wherein said candidate agent is a siRNA.