Metformin increased the expression of cell surface DR5 only

empty vector control p < 0.02. C. INS1 832/13 cells were transfected with Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, RNA was collected and rIns2 mRNA was measured by qRT-PCR analysis. Each bar represents relative Ins2 mRNA normalized to 18s rRNA +/- SEM. Indicates statistically different value compared to empty vector control p < 0.02. doi:10.1371/journal.pone.0131303.g008 Discussion Glis3-mediated transcriptional activation is likely regulated at multiple levels, including transcriptional, translational and post-translational mechanisms that control Glis3 protein activity and/or its expression level. We previously reported that the central region of Glis3 containing the ZFD and the C-terminal transactivation domain are essential for Glis3-mediated transcriptional activation. In this study, we discover a new function for the N-terminus of Glis3 in the regulation of Glis3 stability and its transcriptional activity. Using the N-terminus of Glis3 as bait, we identified by GeLC-MS and Y2H analyses a number of novel Glis3 interacting partners, including several members of the Nedd4/Rsp5 family of HECT E3 ubiquitin ligases. We demonstrate that the HECT E3 ubiquitin ligase, Itch, promotes the polyubiquitination of Glis3, thereby targeting Glis3 for increased proteolytic degradation by the proteasome. Consequently, this leads to a substantially reduced Glis3 transcriptional activity as indicated by the decrease in Glis3-mediated activation of the mIns2 promoter as well as the transactivation of a reporter PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19744531 driven by 3xGlisBS. Itch and other E3 ligases have been reported to regulate the activity of various transcription factors and biological processes through different mechanisms. For example, increased ubiquitination of p73 by Itch has been demonstrated to promote its degradation and consequently its transcriptional activity and function, while the hedgehog transcription factor Gli1 is targeted by Numb PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740905 for Itch-dependent ubiquitination thereby inhibiting growth and promoting cell differentiation. Itch has also been reported to interact with and target the pluripotency-associated transcription factor Oct4 for ubiquitination thereby affecting embryonic stem cell self renewal. The inhibition of Glis3 activity by Itch was dependent on the interaction of Glis3 with and its ubiquitination by Itch since Itch had little effect on the transcriptional activity of the Glis3 mutant in which the Itch-interaction motif was mutated. Moreover, a catalytically inactive Itch mutant had little effect on Glis3-mediated transactivation. These observations are consistent with the conclusion that the reduction in transcriptional activity of Glis3 by Itch was likely Cobicistat related to decreased levels of Glis3 protein. Similarly, overexpression of Itch, but not the catalytically inactive mutant, in rat insulinoma INS1 832/13 cells resulted in a decrease in 15 / 22 Regulation of Glis3 Activity by the HECT E3 Ubiquitin Ligases endogenous rIns2 mRNA expression presumably due to proteolytic degradation of endogenous Glis3. It is tempting to speculate that this regulation of Glis3 protein levels by Itch could serve as a mechanism for fine-tuning Glis3 function in pancreatic beta cells and might be part of the transcriptional control of insulin gene expression. In this context, it is interesting to note that heterozygous Glis3 null mice, which presumably express half the amount of Glis3, are more susceptible to developing glucose intolerance supporting th