Effect of anabolic agents on calpastatin promoters in porcine skeletal muscle and their responsiveness to cyclic adenosine monophosphate-and calcium-related stimuli

• Published in: Journal of animal science Vol. 84; no. 11; pp. 2973 - 2982
• Main Authors: Sensky, P.L, Jewell, K.K, Ryan, K.J.P, Parr, T, Bardsley, R.G, Buttery, P.J
• Format: Journal Article
• Language: English
• Published: United States American Society of Animal Science 01.11.2006; Am Soc Animal Sci
• Subjects: Anabolic Agents - pharmacology; Animals; beta-adrenergic agonists; binding sites; calcineurin; calcium; Calcium - metabolism; Calcium - pharmacology; Calcium-Binding Proteins - genetics; calpain; calpastatin; Cell Line; clenbuterol; Clenbuterol - pharmacology; cyclic AMP; Cyclic AMP - pharmacology; cyclosporins; exons; Gene Expression Regulation - drug effects; Glycogen - metabolism; growth promotion; in vitro studies; ionophores; mechanism of action; molecular sequence data; Muscle, Skeletal - drug effects; Muscle, Skeletal - metabolism; polymerase chain reaction; promoter regions; Promoter Regions, Genetic - genetics; reporter genes; RNA, Messenger; signal transduction; skeletal muscle; swine; Swine - metabolism; transcription factors
• ISSN: 0021-8812; 1525-3163
• DOI: 10.2527/jas.2006-073

The calpain proteinases and their specific inhibitor calpastatin have been proposed to influence both the rates of myofibrillar protein turnover in vivo and meat tenderization postmortem. Elevated calpastatin concentrations in particular are associated with certain forms of hypertrophic growth and meat toughness. In the 5'region of the porcine calpastatin gene, there are 3 calpastatin promoters upstream of exons 1xa, 1xb, and 1u, respectively, each of which contain transcription factor-binding motifs, suggesting sensitivity to a variety of growth-promoting stimuli. This study examined the effect of the β-adrenergic agonist clenbuterol and porcine ST (pST) treatment on calpastatin promoter usage in porcine LM in vivo using real-time PCR and also the responsiveness of transfected calpastatin promoter sequences to cyclic adenosine monophosphate (cAMP) and calcium (Ca²⁺)-related stimuli in reporter gene systems in cell studies. The effect of clenbuterol and pST on potential signaling pathways in vivo was also assessed by monitoring protein phosphatase 2B (calcineurin), NFATc3, calpain 3, IκBα, and NFκB by quantitative immunoblotting. Total calpastatin mRNA was increased by 52% (P < 0.05) after treatment with clenbuterol for 1 d and reduced by 35% (P < 0.01) after pST treatment for 7 d. Whereas clenbuterol had no significant differential effects on individual mRNA transcripts (types 1 to 3) derived from the 3 upstream promoters, pST significantly reduced all of these by 51, 39, and 40% (P < 0.001, 0.05, and 0.05), respectively. Promoter activity was increased in rat L6G8 cells transfected with a construct derived from exon 1u after treatment with dibutyryl cAMP (68%, P < 0.05) or forskolin (43%, P < 0.05), whereas 1xa activity was reduced by both of these agents (47 and 33%, respectively, P < 0.05). Treatment of cells with the calcium ionophore calcimycin reduced the activity of the 1u promoter by 40% (P < 0.01), with no effect on the other promoter constructs. Cyclosporin A had no effect on any promoter construct. The only signaling pathway component to be significantly altered by the in vivo treatments was calcineurin, which was decreased by 24% (P < 0.05) in clenbuterol-treated animals. In conclusion, 2 types of growth promoter in pigs had contrasting effects on calpastatin expression in LM. Transfected calpastatin promoters were differentially sensitive to cAMP- and Ca²⁺-related stimuli, in agreement with the proposed mode of action of the 2 growth promoters.