Tumor necrosis factor-α enhances the transcription of Smad ubiquitination regulatory factor 1 in an activating protein-1- and Runx2-dependent manner.

ABSTRACT

Smad ubiquitination regulatory factor 1 (Smurf1) is an E3 ubiquitin ligase that negatively regulates osteoblast differentiation. Although tumor necrosis factor-α (TNF-α) has been shown to increase Smurf1 expression, the details of the regulatory mechanisms remain unclear. Here, we investigated the molecular mechanism by which TNF-α stimulates Smurf1 expression in C2C12 and primary cultured mouse calvarial cells. TNF-α treatment rapidly induced the activation of NF-κB and MAPKs. Smurf1 induction by TNF-α was blocked by the inhibition of JNK or ERK, while the inhibition of NF-κB and p38 MAPK had no effect on Smurf1 induction. TNF-α treatment or c-Jun overexpression enhanced the activity of a luciferase reporter that contained a 2.7 kb mouse Smurf1 promoter sequence. Site-directed mutagenesis of the Smurf1 reporter and chromatin immunoprecipitation analysis demonstrated that the activating protein-1 (AP-1) binding motif at -922 bp on the mouse Smurf1 promoter mediated TNF-α/JNK/AP-1-stimulated Smurf1 transcription. Interestingly, Smurf1 expression was not observed in Runx2-null mouse calvarial cells. When Runx2 was ectopically expressed in these cells, the basal and TNF-α-induced expression of Smurf1 was restored. Overexpression of Runx2 transactivated the Smurf1 promoter in a dose-dependent manner. Reporter and chromatin immunoprecipitation assays demonstrated that the Runx2-binding motif at -202 bp functioned in Runx2-mediated Smurf1 expression. ERK activation by TNF-α treatment or constitutively active MEK1 overexpression increased Smurf1 expression in a Runx2-dependent manner. These results suggest that the JNK/AP-1 and ERK/Runx2 signaling pathways mediate TNF-α-dependent Smurf1 transcription.