Identification and Characterization of F‐Box and Leucine‐Rich Repeat Protein 22 (Fbxl22) in Skeletal Muscle

Skeletal muscle atrophy is a physiological condition that results in decreased muscle size and strength and occurs in response to immobilization, denervation, spinal cord injury and aging. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed that F‐Box and Leucine‐Rich Protein 22 (Fbxl22), which has previously been shown to act as an E3 ubiquitin ligase that targets sarcomeric proteins for degradation by the 26S proteasome in cardiac muscle, is also expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that Fbxl22 is expressed in muscle cells, we cloned a novel Fbxl22 intra‐exonic splice variant that codes for a 193 amino acid polypeptide. Additionally, quantitative PCR (qPCR) was used to assess Fbxl22 expression levels in both proliferating and differentiated muscle cells and the results demonstrated that Fbxl22 expression levels are relatively low in proliferating myoblasts, but show significantly elevated expression in differentiated myotubes. In order to characterize the transcriptional regulation of Fbxl22, fragments of the proximal promoter located immediately upstream of the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C 2 C 12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Furthermore, conserved E‐box elements in the proximal promoter region of the Fbxl22 gene were identified, mutated, and analyzed for their role in the transcriptional regulation of Fbxl22 reporter gene activity. The mutation of the conserved E‐box sequences rendered the Fbxl22 reporter genes inactive, suggesting that these elements are potentially necessary for proper Fbxl22 expression in muscle tissue. The discovery that Fbxl22 is expressed in muscle combined with the observation that this gene, a potential E3 ubiquitin ligase, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting. Support or Funding Information The work was support by University of North Florida Transformational Learning Opportunity grants to D.W. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .