Prolyl hydroxylase EGLN3 regulates skeletal myoblast differentiation through an NF-kappaB-dependent pathway.

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.