Jackie M. Wilbraham, Viienne Worral and Brian R. Holloway. Cardiovascular, Metabolism & Musculoskeletal. Research Department, ZENECA Pharmaceuticals,.
666th Meeting Sheffield Held at the University of Sheffield 29-31 July 1997
Biochemical Society Transactions (1998) 26 4
Increased expression of several components of dependant proteosome pathway in denervated rat muscle is reversed by 02adrenergic agonirtr.
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Ian D. Waddell, Pam Ruddock, Katy J. Brocklehurst, Jackie M. Wilbraham, Viienne Worral and Brian R. Holloway
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Cardiovascular, Metabolism & Musculoskeletal Research Department, ZENECA Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire. SK104TG U.K. The ATP dependent pathway has an important role in the degradation of mis-folded or mutant proteins. Recent evidence suggests that it selectively degrades intact cellular proteins, and thereby fulfils important roles in cellular regulation [l]. The role of ubiquitin-dependent proteolysis in the degradation of oncoproteins, tumour suppressers, in cell cycle control, in stress response and the immune system has become increasingly evident over the last few years [2]. What is less clear, however, is the role of ATP ubiquitindependent proteolysis in skeletal muscle loss that occurs during cachexia. Increased activity of the ATP ubiquitindependent proteolysis system in muscle breakdown has been demonstrated in atrophying rat muscle where increased expression of poly-ubiquitin and proteosome mRNA's has been observed [3] . Similar results have been observed in the muscles isolated from tumour bearing rats, in models of sepsis and in humans suffering from severe head trauma and head and neck cancer. Recent studies have confirmed that the ATP ubiquitin-dependent proteolysis system is involved in the degradation of the major myofibrillar components [5]. 02-adrenergic agonists are potent anabolic agents that may act by regulating the ATP ubiquitin-dependent proteolysis system (51. We have identified changes in expression at mRNA and protein levels in the ubiquitinylation enzymes ; E1[6], bendless and UBE2G [8] both skeletal muscle specific E2's and the proteosome component S5a [9] after clenbuterol treatment of rats post unilateral denervation. We also present data on the changes in expression of the MRF family of myogenic differentiation factors including Id1, the expression of which may be regulated by S5a[l0]. Rat soleus muscle total mRNA was prepared using TRI reagent (SIGMA) as per manufacturers instructions (Sigma Technical Bulletin MB-205). 20 mg total RNA samples were electrophoresed on 1o/b denaturing formaldehyde agarose gels in MOPS buffer and transferred onto hybond N' (Amersham). Probes were labelled with =P by random hexamer priming (Amersham), and hybridisations were carried out in QuikHyb hybridization solution (Stratagene) at 65'C as per manufacturers instructions. To control for loading variations, blots were stripped after autoradiography by boiling in O.l%SDS, and then rehybridised using a probe containing 1.2kb of a rat glyceraldehyde-3-phosphate dehydrogenase cDNA (GAPDH) [l 11. Blots were visualised on a Bio-Rad GS-525 Molecular Imager System and the quantity of individual bands assessed by Molecular Analyst v2.1.2 software on a Power Macintosh 8500/180. Results are expressed as a ratio a ~ i r a r yunits for a specific mRNA v GAPDH.
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figure 1 Figure1 :-Changes in levels of mRNA's in innervated and denervated soleus muscle. For panels a-f the following key applies : 1, Control Innervated, 2, Control Denervated, 3, Clenbuterol Innervated and 4, Clenbuterol Denervated. Treatment (clenbuterol 2mg.kg"). began 24 hrs post unilateral denervation. Soleus muscles removed after 4 days of treatment. 1. Hass, A.L. and Siepmann, T.J. (1997) FASEB J 11, 1257. 2. Hilt, W. &Wolf, D. (1996), Trends BiolSci. 21, 96-102 3. Medina R., Wing S. & Goldberg A., (1995), Biochem. J. 307,631 4. Solomeon, V. & Goldberg, A.L., (1996), J. Biol. Chem. 271,26690 5. Costelli,P., Garcia-Martinez,C., Llovera,M., Carbo,N., Lopez-Soriano, F.J., Agell,N., Tessitore,L., Baccin0,F.M. and Argiles, J.M. (1995) J. Clin. Invest. 95 2367-2372 6. Stephen,A., Trausch-Azar,J.S., Ciechanover,A. and Schwartz, A.L. (1996) J. Biol. Chem. 271 15608-15614 7. Yamaguchi,Y, Kim,N.S., Sekine,S., ,Seino,H., Osaka,F., Yamao,F. And KatoS. (1996) J. Biochem (Jap) 120, 494497 8. Watanab0,T.K. Kawai,A. Fujiwara,T. Maekawa,h. Hirai,Y. Nakamura,Y. And Takahashi,E. (1996) Cytogenet Cell Genet 74 146-148 9. Deveraux, Q., van Nocker, S., Mahaffey D., Vierstra R. and Rechsteiner M (1995) J. Biol. Chem. 271 29660-29663 lO.Anand,G., Yin,X., Shahidi,A., Grove,L. and Prochownik, E.V.(1997) J. Biol. Chem. 272 19140-19151 11. Fort,P., Marty,L., Pieechaczyk,M., El Sabrouty,S., Jeanteur,P. and Blanchard,J.M. (1985) Nucleic Acid Res 13 1431-1442
