The Molecular Mechanism of Bcl‐3 Co‐transactivation in Skeletal Muscle Atrophy

The crucial role of NF‐kappaB (Rel) transcription factors is widely appreciated in gene regulation and the associated pathophysiology of many tissues. While much work has been done on individual Rel proteins, one co‐transactivator, Bcl‐3, has been underappreciated in the extent of its participation in NF‐kappaB‐dependent disease. Previously we showed that Bcl‐3 is required for the activation of a NF‐kappaB reporter during skeletal muscle atrophy. Without Bcl‐3, inactivity does not produce atrophy and fails to induce several genes whose promoters show Bcl‐3 binding by ChIP‐sequencing, including MuRF1 and Ubr1, two genes whose protein products participate in muscle proteolysis. There was low level activity of promoter‐reporter plasmids for NF‐κB, MuRF1 and Ubr1 in Bcl3‐/‐ fibroblasts but all reporters showed increased activity when the cells were co‐transfected with a plasmid expressing Bcl‐3. To further investigate the role of Bcl‐3 in NF‐κB activation and muscle atrophy, we have constructed a dominant negative form of Bcl‐3 by deleting its two transactivation domains. Co‐transfection experiments using a plasmid coding for an EGFP fusion version of this mutant Bcl‐3 show that it is effective at blocking the activation of an NF‐κB reporter induced by the combination of wild type Bcl‐3 and p52 in myotubes; the latter is a Rel protein that provides DNA binding for the active complex of p52 and the Bcl‐3 co‐transactivator. This inhibition is accompanied by accumulation of green fluorescence from the dominant negative Bcl‐3 mutant in the nuclei of co‐transfected myotubes. Using this new Bcl‐3 mutant we have begun to study the formation of the active NF‐κB complex in myotubes and how the specific inhibition of Bcl‐3 in adult muscle affects the activity of NF‐κB and the activation of genes that carry out muscle wasting.