Smith M A, Fanger G R, O'Connor L T, Bridle P, Maue R A
Department of Anatomy and Neurobiology, University of California at Irvine, Irvine, California 92697, USA.
J Biol Chem. 1997 Jun 20;272(25):15675-81. doi: 10.1074/jbc.272.25.15675.
The extracellular matrix protein agrin plays an important role in the formation and maintenance of the neuromuscular junction. However, regulation of agrin gene expression and pre-mRNA splicing, important in determining the biological actions of agrin, is not well understood. To begin to identify mechanisms controlling agrin expression, quantitative polymerase chain reaction techniques were used to analyze the effect of growth factors on the expression of agrin mRNA isoforms in rat pheochromocytoma (PC12) cells. Agrin transcripts in untreated cells lacked inserts in the Y and Z sites (agriny0z0), encoding agrin isoforms with low acetylcholine receptor aggregating activity and a primarily non-neuronal tissue distribution. Transcripts encoding isoforms with high aggregating activity and neuronal tissue distribution (agriny4z8, agriny4z11, and agriny4z19) were not detected. Treatment of PC12 cells with nerve growth factor (NGF) caused a significant increase in total agrin mRNA. In contrast, exposure to epidermal growth factor had no effect. Analysis of alternative splicing of agrin mRNA revealed that NGF elicited a specific increase in agriny4 and agrinz8 mRNAs that did not occur in the presence of epidermal growth factor, insulin, dexamethasone, or retinoic acid. Analysis of PC12 sublines stably overexpressing a dominant inhibitory form of p21 Ras indicated that NGF induced changes in levels of agrin mRNA and alternative splicing required Ras activity. The results show that NGF can influence important aspects of neuronal differentiation by regulating alternative splicing. Furthermore, these data provide insight into the mechanisms governing agrin gene expression and suggest that neurotrophic factors may play a role in regulating agrin expression in vivo.
细胞外基质蛋白聚集蛋白聚糖在神经肌肉接头的形成和维持中起重要作用。然而，聚集蛋白聚糖基因表达和前体mRNA剪接的调控，这对于确定聚集蛋白聚糖的生物学作用很重要，但目前尚未完全了解。为了开始确定控制聚集蛋白聚糖表达的机制，采用定量聚合酶链反应技术分析生长因子对大鼠嗜铬细胞瘤（PC12）细胞中聚集蛋白聚糖mRNA亚型表达的影响。未处理细胞中的聚集蛋白聚糖转录本在Y和Z位点缺乏插入片段（聚集蛋白聚糖y0z0），编码具有低乙酰胆碱受体聚集活性且主要分布于非神经组织的聚集蛋白聚糖亚型。未检测到编码具有高聚集活性和神经组织分布的亚型（聚集蛋白聚糖y4z8、聚集蛋白聚糖y4z11和聚集蛋白聚糖y4z19）的转录本。用神经生长因子（NGF）处理PC12细胞导致聚集蛋白聚糖总mRNA显著增加。相比之下，暴露于表皮生长因子则没有影响。对聚集蛋白聚糖mRNA可变剪接的分析表明，NGF引起聚集蛋白聚糖y4和聚集蛋白聚糖z8 mRNA的特异性增加，而在表皮生长因子、胰岛素、地塞米松或视黄酸存在的情况下则不会发生这种情况。对稳定过表达p21 Ras显性抑制形式的PC12亚系的分析表明，NGF诱导的聚集蛋白聚糖mRNA水平变化和可变剪接需要Ras活性。结果表明，NGF可通过调节可变剪接影响神经元分化的重要方面。此外，这些数据为控制聚集蛋白聚糖基因表达的机制提供了见解，并表明神经营养因子可能在体内调节聚集蛋白聚糖表达中发挥作用。
