Saini Amarjit, Al-Shanti Nasser, Stewart Claire
Institute for Biomedical Research into Human Movement and Health, Manchester Metropolitan University, Manchester, M1 5GD, UK.
Cell Physiol Biochem. 2010;25(2-3):253-62. doi: 10.1159/000276559. Epub 2010 Jan 12.
IGF-I positively impacts on muscle anabolism/regeneration. Using C2 skeletal myoblasts, we previously reported high dose TNF-alpha -induced (10 ng.ml(-1)) cell death is rescued by IGF-I. However, non-myotoxic low dose TNF-alpha (1.25 ng.ml(-1)) elicits a MAPK-mediated apoptotic response when co-incubated with IGF-I (1.5 ng.ml(-1)). Our aim was to investigate these conflicting roles of IGF-I in our model. Insulin array and qRT-PCR identified Adra1d as a potential regulatory gene that was up-regulated in survival and down-regulated under apoptotic conditions. TNF-alpha administration (1.25 or 10 ng.ml(-1)) induced significant decreases ( approximately 50% both incubations) in Adra1d expression relative to DM. IGF-I addition to high dose TNF-alpha (10 ng.ml(-1)) induced myoblast survival and matched a significant (P < 0.05) increase in Adra1d expression. By contrast, IGF-I addition to low dose TNF-alpha (1.25 ng.ml(-1)) induced elevated death resulting in a significant (P < 0.05) decline ( approximately 55%) in Adra1d expression. Pre-administration of PD98059 (20 uM), which rescues death induced by co-incubation of low dose TNF-alpha with IGF-I, Adra1d levels were again comparable to DM control. Since Adra1d was elevated following incubations that induced myoblast survival, we investigated effects of Adra1d siRNA gene silencing under these conditions. Adra1d knockdown resulted in significantly higher levels of cell death under all incubations suggesting Adra1d expression is essential for skeletal muscle cell survival.
胰岛素样生长因子-I（IGF-I）对肌肉合成代谢/再生有积极影响。我们之前使用C2骨骼肌成肌细胞报道，高剂量肿瘤坏死因子-α（TNF-α）（10 ng·ml⁻¹）诱导的细胞死亡可被IGF-I挽救。然而，与IGF-I（1.5 ng·ml⁻¹）共同孵育时，非肌毒性低剂量TNF-α（1.25 ng·ml⁻¹）会引发丝裂原活化蛋白激酶（MAPK）介导的凋亡反应。我们的目的是在我们的模型中研究IGF-I的这些相互矛盾的作用。胰岛素阵列和定量逆转录聚合酶链反应（qRT-PCR）确定肾上腺素能α-1D受体（Adra1d）为一个潜在的调控基因，该基因在存活条件下上调，在凋亡条件下下调。与糖尿病培养基（DM）相比，给予TNF-α（1.25或10 ng·ml⁻¹）可导致Adra1d表达显著降低（两种孵育条件下均约降低50%）。在高剂量TNF-α（10 ng·ml⁻¹）中添加IGF-I可诱导成肌细胞存活，并使Adra1d表达显著（P < 0.05）增加。相比之下，在低剂量TNF-α（1.25 ng·ml⁻¹）中添加IGF-I会导致死亡增加，从而使Adra1d表达显著（P < 0.05）下降（约55%）。预先给予PD98059（20 μM）可挽救低剂量TNF-α与IGF-I共同孵育诱导的死亡，此时Adra1d水平再次与DM对照相当。由于在诱导成肌细胞存活的孵育后Adra1d升高，我们研究了在这些条件下Adra1d小干扰RNA（siRNA）基因沉默的影响。Adra1d基因敲低导致在所有孵育条件下细胞死亡水平显著更高，表明Adra1d表达对骨骼肌细胞存活至关重要。
