Orzechowski Arkadiusz, Jank Michał, Gajkowska Barbara, Sadkowski Tomasz, Godlewski Michał Marek, Ostaszewski Piotr
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland.
J Muscle Res Cell Motil. 2003;24(1):33-53. doi: 10.1023/a:1024887431768.
The molecular mechanism of the cell death-promoting effect of dexamethasone (Dex) was studied during myogenesis (10 days) in L6 muscle cells by making use of several indices such as cell viability (protein synthesis, mitochondrial respiration), mortality (DNA fragmentation, chromatin condensation, structural modifications) and immunocytochemical studies [hydrogen peroxide, m-calpain (calpain 2)]. Dex initially (2 nM) stimulated protein synthesis (P < 0.001), but a further increase (20 nM) did not stimulate, whereas a higher dose (200 nM) inhibited formation of cellular proteins (P < 0.001). The latter, apparently, resulted from impaired cell viability (P < 0.001). From the day 4, structural changes featuring cell death were observed. Antioxidants [sodium ascorbate (ASC), catalase (CAT) or N-acetyl-L-cysteine (NAC)] as well as the inhibition of transcription and translation by actinomycin D abrogated Dex-induced cell death (P < 0.001). Using a fluorescent probe (DCFH-DA) we directly corroborated the working hypothesis of the mediating role of H2O2 in the reduction of cell viability by the excess of glucocorticoids. We also found that tPKC, PLCgamma, PLA2 were required to induce Dex-dependent cell death since inactivation of tPKC by H7 completely abolished the cytotoxic effect of Dex, while the blockade of PLCgamma and PLA2 by U 73122 partially abolished the effect. Cell death was triggered by Ca2+ influx necessary to activate m-calpain since it was reversed by the calcium chelator EGTA or m-calpain inhibitor ALLN but not EDTA nor ALLM. However, cell viability impaired by Ca2+ ionophore A 23187 (P < 0.001) was neither reversed by EGTA, nor EDTA, nor caspase-3 blocker--Ac DEVD CHO, nor ALLN, nor antioxidants--ASC, NAC, CAT. Specific caspase-3 inhibitor Ac DEVD CHO also did not rescue cells from Dex-induced cell death (P < 0.001), in contrast to m-calpain inhibitor--ALLN. Taken together, these findings suggest that reactive oxygen species inhibit protein synthesis and amplify m-calpain-dependent proteolysis. The events that led to the death of L6 muscle cells most likely resulted from Dex-mediated repression of antioxidative defences on the genomic level.
通过利用细胞活力（蛋白质合成、线粒体呼吸）、死亡率（DNA片段化、染色质凝聚、结构改变）以及免疫细胞化学研究[过氧化氢、m-钙蛋白酶（钙蛋白酶2）]等多项指标，研究了地塞米松（Dex）在L6肌肉细胞成肌过程（10天）中促进细胞死亡的分子机制。Dex最初（2 nM）刺激蛋白质合成（P<0.001），但进一步增加（20 nM）时并无刺激作用，而更高剂量（200 nM）则抑制细胞蛋白质的形成（P<0.001）。后者显然是由于细胞活力受损（P<0.001）所致。从第4天起，观察到了以细胞死亡为特征的结构变化。抗氧化剂[抗坏血酸钠（ASC）、过氧化氢酶（CAT）或N-乙酰-L-半胱氨酸（NAC）]以及放线菌素D对转录和翻译的抑制作用消除了Dex诱导的细胞死亡（P<0.001）。使用荧光探针（DCFH-DA），我们直接证实了过氧化氢在过量糖皮质激素降低细胞活力过程中起介导作用的工作假设。我们还发现，tPKC、PLCγ、PLA2是诱导Dex依赖性细胞死亡所必需的，因为H7使tPKC失活完全消除了Dex的细胞毒性作用，而U 73122对PLCγ和PLA2的阻断部分消除了该作用。细胞死亡是由激活m-钙蛋白酶所需的钙离子内流触发的，因为它可被钙螯合剂EGTA或m-钙蛋白酶抑制剂ALLN逆转，但不能被EDTA或ALLM逆转。然而，钙离子载体A 23187损害的细胞活力（P<0.001）既不能被EGTA、EDTA、半胱天冬酶-3阻断剂——Ac DEVD CHO、ALLN逆转，也不能被抗氧化剂——ASC、NAC、CAT逆转。与m-钙蛋白酶抑制剂——ALLN相反，特异性半胱天冬酶-3抑制剂Ac DEVD CHO也不能使细胞从Dex诱导的细胞死亡中获救（P<0.001）。综上所述，这些发现表明活性氧抑制蛋白质合成并放大m-钙蛋白酶依赖性蛋白水解。导致L6肌肉细胞死亡的事件很可能是由Dex在基因组水平上对抗氧化防御的抑制作用引起的。
