The Regulation of C/EBPβ Activity and Adipogenesis by the Smad3 MH1 Domain

Abstract

Retinoic Acid (RA) is a potent anti-adipogenic molecule. Recent experiments in our lab identified the transcription factor Smad3 as a novel RA target gene required for RA-mediated inhibition of adipogenesis. Smad3 was demonstrated to inhibit C/EBPβ DNA binding via an interaction between the Smad3-MH1 domain and C/EBPβ. The goal of this thesis was to evaluate the anti-adipogenic potential of the isolated Smad3 MH1 domain in the absence of RA treatment. Pooled 3T3-L1 preadipocyte stable cell lines expressing MH1 and empty vector controls were created and induced to differentiate. While MH1 protein expression was difficult to detect due to high protein lability, C/EBPβ DNA binding to its response element within the C/EBPα promoter was inhibited in MH1-expressing cells. Despite this, loss of C/EBPβ occupancy at the C/EBPα promoter did not result in changes in C/EBPα gene expression though adipogenesis was inhibited in these cultures. In contrast, PPARγ levels were reduced in MH1expressing cells suggesting that regulation of PPARγ expression by the isolated MH1 domain drives the inhibition of adipogenesis in this model. The second goal of this thesis was to evaluate the regulation of Smad3 by RA. We determined that RA directly activates Smad3 transcription, which is not dependent on promoter demethylation. Furthermore, RA induces Smad3 nuclear accumulation in the absence of Smad3 phosphorylation.