IGF-I-induced differentiation of L6 myogenic cells requires the activity of cAMP-phosphodiesterase
IGF-I-induced differentiation of L6 myogenic cells requires the activity of cAMP-phosphodiesterase
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DOI：
10.1091/mbc.e02-03-0156
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发表时间：
2003-04-01
影响因子：
3.3
通讯作者：
Naro, F
中科院分区：
文献类型：
--
作者：
De Arcangelis, V;Coletti, D;Naro, F
抑制 L6-C5 和 L6-E9 中 4 型 cAMP 特异性磷酸二酯酶 (PDE4) 活性可消除低血清培养基和 IGF-I 诱导的肌原性分化。在低血清培养基中培养的L6-C5细胞显示出比在无血清培养基中培养的细胞更高的PDE4活性，无血清培养基是不足以诱导分化的条件。在血清存在的情况下，L6-C5 细胞中天然表达的主要亚型 PDE4D3 转位至 Triton 不溶性级分，这增加了该级分的 PDE 比活性，并表现出该亚型磷酸化的典型 Mr 转变。此外，血清促进 PDE4D3 定位到囊泡亚细胞区室。在 L6-C5 细胞中，IGF-I 在存在血清时比在不存在血清时是更强的肌原性分化诱导剂。通过过表达野生型 PDE4D3（而非磷酸化不敏感突变体）可以恢复其在缺乏血清的情况下触发分化的能力。通过评估 L6-C5 和 L6-E9 中肌细胞生成素的核积累，在过表达 GFP-PDE4D3 融合蛋白的单细胞中证实了这一发现。其他 PDE 同工型的过度表达效率较低，这证实了 PDE4D3 是控制肌生成的生理相关磷酸二酯酶同工型。这些结果表明，通过 cAMP 磷酸二酯酶刺激下调 cAMP 信号传导是诱导肌生成的先决条件。
Inhibition of type 4 cAMP-specific phosphodiesterase (PDE4) activity in L6-C5 and L6-E9 abolished myogenic differentiation induced by low-serum medium and IGF-I. L6-C5 cells cultured in low-serum medium displayed a PDE4 activity higher than cells cultured in serum-free medium, a condition not sufficient to induce differentiation. In the presence of serum, PDE4D3, the major isoform natively expressed in L6-C5 cells, translocated to a Triton-insoluble fraction, which increased the PDE specific activity of the fraction, and exhibited a Mr shift typical of phosphorylation of this isoform. Furthermore, serum promoted the localization of PDE4D3 to a vesicular subcellular compartment. In L6-C5 cells, IGF-I is a stronger inducer of myogenic differentiation in the presence than in absence of serum. Its ability to trigger differentiation in the absence of serum was restored by overexpressing wild-type PDE4D3, but not a phosphorylation-insensitive mutant. This finding was confirmed in single cells overexpressing a GFP-PDE4D3 fusion protein by assessing nuclear accumulation of myogenin in both L6-C5 and L6-E9. Overexpression of other PDE isoforms was less efficient, confirming that PDE4D3 is the physiologically relevant phosphodiesterase isoform in the control of myogenesis. These results show that downregulation of cAMP signaling through cAMP-phosphodiesterase stimulation is a prerequisite for induction of myogenesis.