Wyke S M, Tisdale M J
Biomedicinal Chemistry Research Group, School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK.
Life Sci. 2006 May 15;78(25):2898-910. doi: 10.1016/j.lfs.2005.11.014. Epub 2005 Dec 15.
Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose-response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 microM), suggesting that it was mediated through the ubiquitin-proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin-proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome alpha-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E2(14k), also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin-proteasome pathway. TPA also induced degradation of the inhibitory protein, I-kappaBalpha, and increased nuclear accumulation of nuclear factor-kappaB (NF-kappaB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-kappaB activation by resveratrol (30 microM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-kappaB.
尽管肌肉萎缩在多种疾病状态中都很常见，但对于其中涉及的细胞机制我们了解并不完全。在本研究中，用佛波酯12 - O - 十四酰佛波醇 - 13 - 乙酸酯（TPA）处理小鼠肌管，以评估蛋白激酶C（PKC）作为蛋白质降解上游中间体的作用。TPA对总蛋白降解的诱导呈现抛物线剂量反应曲线，在浓度为25 nM时效果最佳，最佳孵育时间为3小时。与蛋白酶体抑制剂乳胞素（5 microM）共同孵育可减弱蛋白质降解，这表明其是通过泛素 - 蛋白酶体蛋白水解途径介导的。TPA诱导泛素 - 蛋白酶体途径的表达和活性增加，表现为功能活性增强，20S蛋白酶体α亚基、19S亚基MSS1和p42以及泛素缀合酶E2(14k)的表达增加，同样在浓度为25 nM和孵育3小时时效果最佳。肌原纤维蛋白肌球蛋白的细胞含量也呈相应下降。TPA在浓度为25 nM时最大程度地诱导PKC活化，这种作用被PKC抑制剂钙泊三醇C（300 nM）减弱，总蛋白降解也如此。这些结果表明，肌肉细胞中PKC的刺激通过泛素 - 蛋白酶体途径启动蛋白质降解。TPA还在诱导蛋白酶体表达的相同时间和浓度下诱导抑制蛋白I - κBα的降解，并增加核因子κB（NF - κB）的核内积累。此外，白藜芦醇（30 microM）对NF - κB活化的抑制减弱了TPA诱导的蛋白质降解。这些结果表明，TPA对蛋白酶体表达的诱导可能涉及转录因子NF - κB。
