Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
J Biol Chem. 2010 Mar 19;285(12):8927-35. doi: 10.1074/jbc.M109.078600. Epub 2010 Jan 10.
The egg-laying abnormal-9 (EGLN) prolyl hydroxylases have been shown to regulate the stability and thereby the activity of the alpha subunits of hypoxia-inducible factor (HIF) through its ability to catalyze their hydroxylation. We have previously shown that EGLN3 promotes differentiation of C2C12 skeletal myoblasts. However, the mechanism underlying this effect remains to be fully elucidated. Here, we report that exposure of C2C12 cells to dimethyl oxalylglycine (DMOG), desferrioxamine, and hypoxia, all inhibitors of prolyl hydroxylase activity, led to repression of C2C12 myogenic differentiation. Inactivation of HIF by expression of a HIF dominant-negative mutant or deletion of HIF-1alpha by RNA interference did not affect the inhibitory effect of DMOG, suggesting that the effect of DMOG is HIF-independent. Pharmacologic inactivation of EGLN3 hydroxylase resulted in activation of the canonical NF-kappaB pathway. The inhibitory effect of DMOG on myogenic differentiation was markedly impaired in C2C12 cells expressing a dominant-negative mutant of IkappaBalpha. Exogenous expression of wild-type EGLN3, but not its catalytically inactive mutant, significantly inhibited NF-kappaB activation induced by overexpressed TRAF2 or IkappaB kinase 2. In contrast, deletion of EGLN3 by small interfering RNAs led to activation of NF-kappaB. These data suggest that EGLN3 is a negative regulator of NF-kappaB, and its prolyl hydroxylase activity is required for this effect. Furthermore, wild-type EGLN3, but not its catalytically inactive mutant, potentiated myogenic differentiation. This study demonstrates a novel role for EGLN3 in the regulation of NF-kappaB and suggests that it is involved in mediating myogenic differentiation, which is HIF-independent.
EGLN 家族的脯氨酰羟化酶能够通过催化其羟化反应来调节缺氧诱导因子 (HIF) 的α亚基的稳定性，从而调节其活性。我们之前已经证明，EGLN3 可促进 C2C12 成肌细胞的分化。然而，这种作用的机制仍有待充分阐明。在这里，我们报告说，C2C12 细胞暴露于二甲基草酰甘氨酸 (DMOG)、去铁胺和低氧，所有这些都是脯氨酰羟化酶活性的抑制剂，导致 C2C12 成肌分化受到抑制。表达 HIF 显性负突变体或通过 RNA 干扰删除 HIF-1α 使 HIF 失活并不影响 DMOG 的抑制作用，这表明 DMOG 的作用是不依赖 HIF 的。EGLN3 羟化酶的药理学失活导致经典 NF-κB 途径的激活。在表达 IkappaBalpha 显性负突变体的 C2C12 细胞中，DMOG 对成肌分化的抑制作用明显受损。外源性表达野生型 EGLN3，但不是其催化失活突变体，可显著抑制过表达的 TRAF2 或 IkappaB 激酶 2 诱导的 NF-κB 激活。相反，通过小干扰 RNA 敲低 EGLN3 导致 NF-κB 激活。这些数据表明，EGLN3 是 NF-κB 的负调节剂，其脯氨酰羟化酶活性是必需的。此外，野生型 EGLN3，但不是其催化失活突变体，增强了成肌分化。本研究证明了 EGLN3 在 NF-κB 调节中的新作用，并表明它参与介导与 HIF 无关的成肌分化。
